Carbohydrate conjugates as delivery agents for oligonucleotides

ABSTRACT

The present invention provides iRNA agents comprising at least one subunit of the formula (I): 
     
       
         
         
             
             
         
       
         
         
           
             wherein: 
             A and B are each independently for each occurrence O, N(R N ) or S; 
             X and Y are each independently for each occurrence H, OH, a hydroxyl protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, a polymer, —P(Z′)(Z″)O-Linker-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, an oligonucleotide, —P(Z′)(Z″)-formula(I), —P(Z′)(Z″)— or -Linker-R; 
             R is L G , -Linker-L G , or has the structure shown below: 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             L G  is independently for each occurrence a carbohydrate, e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide; 
             R N  is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; and 
             Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/329,540, filed Jul. 11, 2014, which is a continuation of U.S.application Ser. No. 13/693,478, filed Dec. 4, 2012, now U.S. Pat. No.8,828,956, which is a continuation of U.S. application Ser. No.13/326,203, filed Dec. 14, 2011, now U.S. Pat. No. 8,450,467, which is acontinuation application of U.S. application Ser. No. 12/328,528, filedDec. 4, 2008, now U.S. Pat. No. 8,106,022, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 60/992,309, filedDec. 4, 2007; U.S. Provisional Patent Application No. 61/013,597 fieldDec. 13, 2007; U.S. Provisional Patent Application No. 61/127,751, filedMay 14, 2008; U.S. Provisional Patent Application No. 61/091,093, filedAug. 22, 2008; and U.S. Provisional Patent Application No. 61/097,261,filed Sep. 16, 2008; all of which are herein incorporated by referencein their entirety.

FIELD OF INVENTION

The present invention relates to the field of therapeutic agent deliveryusing carbohydrate conjugates. In particular, the present inventionprovides novel carbohydrate conjugates and iRNA agents comprising theseconjugates, which are advantageous for the in vivo delivery of theseiRNA agents, as well as iRNA compositions suitable for in vivotherapeutic use. Additionally, the present invention provides methods ofmaking these compositions, as well as methods of introducing these iRNAagents into cells using these compositions, e.g., for the treatment ofvarious disease conditions.

BACKGROUND

Oligonucleotide compounds have important therapeutic applications inmedicine. Oligonucleotides can be used to silence genes that areresponsible for a particular disease. Gene-silencing prevents formationof a protein by inhibiting translation. Importantly, gene-silencingagents are a promising alternative to traditional small, organiccompounds that inhibit the function of the protein linked to thedisease. siRNA, antisense RNA, and micro-RNA are oligonucleotides thatprevent the formation of proteins by gene-silencing.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression (Fire et al. (1998) Nature 391, 806-811;Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directsgene-specific, post-transcriptional silencing in many organisms,including vertebrates, and has provided a new tool for studying genefunction. RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.

siRNA compounds are promising agents for a variety of diagnostic andtherapeutic purposes. siRNA compounds can be used to identify thefunction of a gene. In addition, siRNA compounds offer enormouspotential as a new type of pharmaceutical agent which acts by silencingdisease-causing genes. Research is currently underway to developinterference RNA therapeutic agents for the treatment of many diseasesincluding central-nervous-system diseases, inflammatory diseases,metabolic disorders, oncology, infectious diseases, and ocular disease.

siRNA has been shown to be extremely effective as a potential anti-viraltherapeutic with numerous published examples appearing recently. siRNAmolecules directed against targets in the viral genome dramaticallyreduce viral titers by orders of magnitude in animal models of influenza(Ge et al., (2004) Proc. Natl. Acd. Sci. USA, 101, 8676-8681; Tompkinset al. (2004) Proc. Natl. Acd. Sci. USA, 101, 8682-8686; Thomas et al.(2005) Expert Opin. Biol. Ther. 5, 495-505), respiratory synctial virus(RSV) (Bitko et al. (2005) Nat. Med. 11, 50-55), hepatitis B virus (HBV)(Morrissey et al. (2005) Nat. Biotechnol. 23, 1002-1007), hepatitis Cvirus (Kapadia et al. (2003) Proc. Natl. Acad. Sci. USA, 100, 2014-2018;Wilson et al. (2003) Proc. Natl. Acad. Sci. USA, 100, 2783-2788) andSARS coronavirus (Li et al. (2005) Nat. Med. 11, 944-951).

Antisense methodology is the complementary hybridization of relativelyshort oligonucleotides to mRNA or DNA such that the normal, essentialfunctions, such as protein synthesis, of these intracellular nucleicacids are disrupted. Hybridization is the sequence-specific hydrogenbonding via Watson-Crick base pairs of oligonucleotides to RNA orsingle-stranded DNA. Such base pairs are said to be complementary to oneanother.

The naturally-occurring events that alter the expression level of thetarget sequence, discussed by Cohen (Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., 1989, Boca Raton, Fla.)are thought to be of two types. The first, hybridization arrest,describes the terminating event in which the oligonucleotide inhibitorbinds to the target nucleic acid and thus prevents, by simple sterichindrance, the binding of essential proteins, most often ribosomes, tothe nucleic acid. Methyl phosphonate oligonucleotides (Miller et al.(1987) Anti-Cancer Drug Design, 2, 117-128), and α-anomeroligonucleotides are the two most extensively studied antisense agentswhich are thought to disrupt nucleic acid function by hybridizationarrest.

Another means by which antisense oligonucleotides alter the expressionlevel of target sequences is by hybridization to a target mRNA, followedby enzymatic cleavage of the targeted RNA by intracellular RNase H. A2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analoghybridizes with the targeted RNA and this duplex activates the RNase Henzyme to cleave the RNA strand, thus destroying the normal function ofthe RNA. Phosphorothioate oligonucleotides are the most prominentexample of an antisense agent that operates by this type of antisenseterminating event.

The opportunity to use these and other nucleic acid based therapiesholds significant promise, providing solutions to medical problems thatcould not be addressed with current, traditional medicines. The locationand sequences of an increasing number of disease-related genes are beingidentified, and clinical testing of nucleic acid-based therapeutics fora variety of diseases is now underway.

Despite the advances in application of oligonucleotides andoligonucleotide analogs as therapeutics, the need exists foroligonucleotides having improved pharmacological properties, e.g. serumstability, delivery to the right organ or cell and transmembranedelivery. Efforts aimed at improving the transmembrane delivery ofnucleic acids and oligonucleotides have utilized protein carriers,antibody carriers, liposomal delivery systems, electroporation, directinjection, cell fusion, viral vectors, and calcium phosphate-mediatedtransformation. However, many of these techniques are limited by thetypes of cells in which transmembrane transport is enabled and by theconditions needed for achieving such transport.

Efficient delivery to cells in vivo requires specific targeting andsubstantial protection from the extracellular environment, particularlyserum proteins. One method of achieving specific targeting is toconjugate a targeting moiety to the iRNA agent. The targeting moietyhelps in targeting the iRNA agent to the required target site. One way atargeting moiety can improve delivery is by receptor mediatedendocytotic activity. This mechanism of uptake involves the movement ofiRNA agent bound to membrane receptors into the interior of an area thatis enveloped by the membrane via invagination of the membrane structureor by fusion of the delivery system with the cell membrane. This processis initiated via activation of a cell-surface or membrane receptorfollowing binding of a specific ligand to the receptor. Manyreceptor-mediated endocytotic systems are known and have been studied,including those that recognize sugars such as galactose, mannose,mannose-6-phosphate, peptides and proteins such as transferrin,asialoglycoprotein, vitamin B12, insulin and epidermal growth factor(EGF). The Asialoglycoprotein receptor (ASGP-R) is a high capacityreceptor, which is highly abundant on hepatocytes. The ASGP-R shows a50-fold higher affinity for N-Acetyl-D-Galactosylamine (GalNAc) thanD-Gal. Previous work has shown that multivalency is required to achievenM affinity, while spacing among sugars is also crucial.

The Mannose receptor, with its high affinity to D-mannose representsanother important carbohydrate-based ligand-receptor pair. The mannosereceptor is highly expressed on specific cell types such as macrophagesand possibly dendritic cells Mannose conjugates as well as mannosylateddrug carriers have been successfully used to target drug molecules tothose cells. For examples, see Biessen et al. (1996) J. Biol. Chem. 271,28024-28030; Kinzel et al. (2003) J. Peptide Sci. 9, 375-385; Barratt etal. (1986) Biochim. Biophys. Acta 862, 153-64; Diebold et al. (2002)Somat. Cell Mol. Genetics 27, 65-74.

Lipophilic moieties, such as cholesterol or fatty acids, when attachedto highly hydrophilic molecules such as nucleic acids can substantiallyenhance plasma protein binding and consequently circulation half life.In addition, binding to certain plasma proteins, such as lipoproteins,has been shown to increase uptake in specific tissues expressing thecorresponding lipoprotein receptors (e.g., LDL-receptor HDL-receptor orthe scavenger receptor SR-B1). For examples, see Bijsterbosch, M. K.,Rump, E. T. et al. (2000) Nucleic Acids Res. 28, 2717-25; Wolfrum, C.,Shi, S. et al. (2007) 25, 1149-57. Lipophilic conjugates can also beused in combination with the targeting ligands in order to improve theintracellular trafficking of the targeted delivery approach.

There is a clear need for new receptor specific ligand conjugated iRNAagents and methods for their preparation, that address the shortcomingsof the in vivo delivery of oligonucleotide therapeutics as describedabove. The present invention is directed to this very important end.

SUMMARY

In one aspect, the invention provides an iRNA agent that is conjugatedwith at least one carbohydrate ligand, e.g., monosaccharide,disaccharide, trisaccharide, tetrasaccharide, oligosaccharide,polysaccharide. These carbohydrate-conjugated iRNA agents target, inparticular, the parenchymal cells of the liver. In one embodiment, theiRNA agent includes more than one carbohydrate ligand, preferably two orthree. In one embodiment, the iRNA agent comprises one or more galactosemoiety. In another embodiment, the iRNA agent includes at least one(e.g., two or three or more) lactose molecules (lactose is a glucosecoupled to a galactose). In another embodiment, the iRNA agent includesat least one (e.g., two or three or more) N-Acetyl-Galactosamine(GalNAc), N—Ac-Glucosamine (GluNAc), or mannose (e.g.,mannose-6-phosphate). In one embodiment, iRNA agent comprises at leastone mannose ligand, and the iRNA agent targets macrophages.

In one aspect, the invention features an iRNA agent comprising acarbohydrate ligand, and the presence of the carbohydrate ligand canincrease delivery of the iRNA agent to the liver. Thus an iRNA agentcomprising a carbohydrate ligand can be useful for targeting a gene forwhich expression is undesired in the liver. For example, an iRNA agentcomprising a carbohydrate ligand can target a nucleic acid expresses bya hepatitis virus (e.g., hepatitis C, hepatitis B, hepatitis A,hepatitis D, hepatitis E, hepatitis F, hepatitis G, or hepatitis H).

In one embodiment, the carbohydrate-conjugated iRNA agent targets a geneof the hepatitis C virus. In another embodiment, the iRNA agent thattargets a gene of the hepatitis C virus can be administered to a humanhaving or at risk for developing hepatitis, e.g., acute or chronichepatitis, or inflammation of the liver. A human who is a candidate fortreatment with a carbohydrate-conjugated iRNA agent, e.g., an iRNA agentthat targets a gene of HCV, can present symptoms indicative of HCVinfection, such as jaundice, abdominal pain, liver enlargement andfatigue.

In one embodiment, a carbohydrate-conjugated iRNA agent targets the 5′core region of HCV. This region lies just downstream of the ribosomaltoe-print straddling the initiator methionine. In another embodiment, aniRNA agent targets any one of the nonstructural proteins of HCV, such asNS3, NS4A, NS4B, NS5A, or NS5B. In another embodiment, an iRNA agenttargets the E1, E2, or C gene of HCV.

In another embodiment, the carbohydrate-conjugated iRNA agent targets ahepatitis B virus (HBV), and the iRNA agent has a sequence that issubstantially similar to a sequence of a gene of HBV, e.g., the proteinX (HBx) gene of HBV.

Carbohydrate-conjugated iRNA agents can also be used to treat otherliver disorders, including disorders characterized by unwanted cellproliferation, hematological disorders, metabolic disorders, anddisorders characterized by inflammation. A proliferation disorder of theliver can be, for example, a benign or malignant disorder, e.g., acancer, e.g, a hepatocellular carcinoma (HCC), hepatic metastasis, orhepatoblastoma. A hepatic hematology or inflammation disorder can be adisorder involving clotting factors, a complement-mediated inflammationor a fibrosis, for example. Metabolic diseases of the liver includedyslipidemias and irregularities in glucose regulation. In oneembodiment, a liver disorder is treated by administering one or moreiRNA agents that have a sequence that is substantially identical to asequence in a gene involved in the liver disorder.

In one embodiment, a carbohydrate-conjugated iRNA agent targets anucleic acid expressed in the liver, such as an ApoB RNA, c-jun RNA,beta-catenin RNA, or glucose-6-phosphatase mRNA.

An iRNA that targets glucose-6-phosphatase can be administered to asubject to inhibit hepatic glucose production, e.g., for the treatmentof glucose-metabolism-related disorders, such as diabetes, e.g.,type-2-diabetes mellitus. The iRNA agent can be administered to anindividual at risk for the disorder to delay onset of the disorder or asymptom of the disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Conjugation of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to 3′-(I) and 5′-ends (II) of doublestranded nucleic acids and 3′-(III) and 5′-ends (IV) of single strandednucleic acids. Double stranded nucleic acids can have two 3′-overhangs,one overhang at 3′-end of sense or antisense or with no overhangs; Q isO or S.

FIG. 2. Conjugation of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to: (V) 3′-ends of both strands (senseand antisense or guide strands); (VI) 3′-end of one strand (sense orantisense) and 5′-end of second the complementary strand and (VII)5′-ends of both strands. Double stranded nucleic acids can have two3′-overhangs, one overhang at 3′-end of sense or antisense or with nooverhangs.

FIG. 3. Conjugation of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to: 3′- and 5′-ends of one strand(sense or antisense/guide strands). Double stranded nucleic acids canhave two 3′-overhangs, one overhang at 3′-end of sense or antisense orwith no overhangs.

FIG. 4. Conjugation of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to: 3′-end (IX) and 5′-end (X) of senseor antisense strand; q=0-10. Double stranded nucleic acids can have two3′-overhangs, one overhang at 3′-end of sense or antisense or with nooverhangs.

FIG. 5. Conjugation of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to: 3′ and 5′-ends (XI); 3′-end (XII)and 5′-end (XIII) of oligonucleotide; q=0-10.

FIG. 6. Conjugation of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to nucleic acids with additional spacerseparation: XIV—3′-end conjugate with alkyl and/or PEG spacer doublestranded nucleic acid; XV—5′-end conjugation with alkyl and/or PEGspacer; XVI and XVII—corresponding 3′ and 5′-end conjugates of singlestranded nucleic acids/oligonucleotides. A, B stands for alkyl or PEGspacer, and combination there of, and Q′=CH₂, O, S, S—S, NH or NMe.Double stranded nucleic acids can have two 3′-overhangs, one overhang at3′-end of sense or antisense or with no overhangs.

FIG. 7. Hybrid conjugates of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc and a second ligand of choice to doublestranded nucleic acids: XX—3′-end serial conjugation; XXI 3′-end ligandand 5′-end pteroic acid analogues on sense or antisense; XXII—5′-endligand and 3′-end pteroic acid analogues on sense or antisense;XXIII—Pteroic acid analogues on 5′-end of sense or antisense and ligandof choice on 3′-end of antisense or sense or vice versa; XXIV—serialconjugation of ligand of choice and pteroic acid analogues to the 5′-endof sense or antisense strand of double stranded nucleic acids. L isligand of choice.

FIG. 8. Hybrid conjugates of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc and a second ligand of choice to singlestranded nucleic acids: XXV—3′- or 5-end serial conjugation; XXVI 3′- or5-end ligand and 5′- or 3-end pteroic acid analogues. L is ligand ofchoice.

FIG. 9. Conjugates of sugars (monosaccharides) to nucleic acids.Conjugation of Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to lipid or lipid like molecule with(XVIII) and without spacer/tether (XIX).

FIG. 10. Conjugation of sugars to nucleic acids. Conjugation oftriantenary Galactose, N-acetylgalactosamine, mannose, glucose,glucosamone, fucose, lactose etc to 3′-(XX) and 5′-ends (XXI) of doublestranded nucleic acids and 3′-(XXII) and 5′-ends (XXIII) of singlestranded nucleic acids. Double stranded nucleic acids can have two3′-overhangs, one overhang at 3′-end of sense or antisense or with nooverhangs; Q is O or S.

FIG. 11. Conjugation of sugars (monosaccharides) to nucleic acids.Conjugation of triantenary Galactose, N-acetylgalactosamine, mannose,glucose, glucosamone, fucose, lactose etc to: (XXIV) 3′-ends of bothstrands (sense and antisense or guide strands); (XXV) 3′-end of onestrand (sense or antisense) and 5′-end of second the complementarystrand and (XXVI) 5′-ends of both strands; For definition of R, X, Y, Zand Q see FIG. 1. Double stranded nucleic acids can have two3′-overhangs, one overhang at 3′-end of sense or antisense or with nooverhangs. Similarly the monoantenary sugar moiety or moieties in FIGS.3-9 are replaced with triantenary sugar moiety or moieties described inFIGS. 10 and 11.

FIG. 12. Conjugation of oligosaccharides to nucleic acids. Conjugationof analogues or derivates of galactose, N-acetylgalactosamine, mannose,glucose, glucosamone, fucose, lactose etc to 3′-(XXVII) and 5′-ends(XXVIII) of double stranded nucleic acids and 3′-(XXIX) and 5′-ends(XXX) of single stranded nucleic acids. Double stranded nucleic acidscan have two 3′-overhangs, one overhang at 3′-end of sense or antisenseor with no overhangs; Q is O or S. Similarly the sugar moiety ormoieties in FIGS. 2-9 are replaced with oligosaccharides moietiesdescribed in FIG. 12.

FIG. 13. Triantenary GalNAc double stranded oligonucleotide conjugateswith cleavable disulfide linkages.

FIG. 14. Triantenary GalNAc double stranded oligonucleotide conjugateswith cleavable disulfide linkages.

FIG. 15. In vivo apoB gene silencing of galactose-siRNA conjugate.

FIG. 16. Structure of cholesterol and (GalNAc)₃ linked together via aphosphate linkage.

FIG. 17. Glycolipid-siRNA conjugate strategies.

FIG. 18. Binding Affinity and Multivalency of the AsialoglycoproteinReceptor.

FIG. 19. Synthesis of multiantennary conjugates from simple monomers.

FIG. 20. Glycolipid-siRNA conjugate for LDL and HDL packing and livertargeting.

FIG. 21. Glycolipid-siRNA Conjugate: Synthesis.

FIG. 22. Monomers for carbohydrate conjugation to siRNA. (A) shows onemonomer for carbohydrate conjugation to siRNA; (B) shows another monomerfor carbohydrate conjugation to siRNA; and (C) shows another monomer forcarbohydrate conjugation to siRNA.

FIG. 23. Synthesis of GalNAc building blocks. (A) shows the synthesis ofone GalNAc building block; (B) shows the synthesis of another GalNAcbuilding block; and (C) shows the synthesis of another GalNAc buildingblock.

FIG. 24. Synthesis of GalNAc building blocks (II).

FIG. 25. GalNAc clusters for hepatic targeting. (A) shows GalNAcconjugate; and (B) shows conjugate of GalNAc clusters.

FIG. 26. Carbohydrate (GalNAC) clusters for conjugation to siRNA.

FIG. 27. Multivalent GalNAC-siRNA conjugates.

FIG. 28. Carbohydrate building blocks for 5′-conjugation.

FIG. 29. Syntheses of Mannose conjugate building blocks.

FIG. 30. Post-synthetic carbohydrate conjugate building blocks.

FIG. 31. Comparison of gene silencing with cholesterol conjugated siRNAversus cholesterol-(GalNAc)3 conjugated siRNA.

FIG. 32. Comparison of duration of effect on serum cholesterol levelswith cholesterol conjugated siRNA versus cholesterol-(GalNAc)3conjugated siRNA.

FIG. 33. Comparison of uptake of Cy3 labeled siRNA with cholesterolconjugated siRNA versus cholesterol-(GalNAc)3 conjugated siRNA.

FIG. 34. Schematic view of design consideration for conjugates.

FIG. 35. Schematic view of designs conjugates.

FIG. 36. Biantennary and Triantennary conjugates.

FIG. 37. Two different conjugates of this invention. (A) shows aconjugate represented by AD-3698 (GalNAc+Chol/Cy3); and (B) shows aconjugate represented by AD-31644 (Q11+L90).

FIGS. 38A-38B. Some exemplary placement of disulfide linkage inconjugates.

FIG. 38A shows a scheme of placement of disulfide linkage in conjugatesas 3′-Chol-S—S-(GalNAc)₃; and FIG. 38B shows a scheme of placement ofdisulfide linkage in conjugates as 3′-S—S-Chol-(GalNAc)₃.

FIGS. 39A-39C. In vivo silencing of FVII with carbohydrate conjugatedsiRNAs. FIG. 39A shows the experimental design for in vivo silencing ofFVII with carbohydrate conjugated siRNAs; FIG. 39B shows the results ofin vivo silencing of FVII with various carbohydrate-conjugated siRNAs;and FIG. 39C shows the results of in vivo silencing of FVII with variouscarbohydrate-conjugated siRNAs.

FIG. 40. In vivo silencing of FVII with carbohydrate conjugated siRNAs.(A) shows the results of in vivo silencing of FVII with variouscarbohydrate-conjugated siRNAs; and (B) shows the results of in vivosilencing of FVII with various carbohydrate-conjugated siRNAs.

FIGS. 41A-41B. In vivo silencing of ApoB with carbohydrate conjugatedsiRNAs. FIG. 41A shows the results of in vivo silencing of ApoB withvarious carbohydrate-conjugated siRNAs samples in liver and jejunum; andFIG. 41B shows the results of total cholesterol in liver.

FIG. 42. In vivo silencing of ApoB with carbohydrate conjugated siRNAs.(A) shows the results of in vivo silencing of ApoB with variouscarbohydrate-conjugated siRNAs samples in liver and jejunum, comparingGalNAc to Glucose; and (B) shows the results of total cholesterol inliver.

FIG. 43. In vitro silencing of ApoB with carbohydrate conjugated siRNAs.

FIG. 44. Competition of carbohydrate conjugated siRNAs with ASGR ligandAsilofetuin (ASF) during in vitro uptake.

FIG. 45. In vitro receptor binding and uptake of carbohydrateconjugates.

FIG. 46. Galactose conjugate (A) and in vivo gene silencing (B).

DETAILED DESCRIPTION

This invention is based on the discovery that conjugation of acarbohydrate moiety to an iRNA agent can optimize one or more propertiesof the iRNA agent. In many cases, the carbohydrate moiety will beattached to a modified subunit of the iRNA agent. E.g., the ribose sugarof one or more ribonucleotide subunits of an iRNA agent can be replacedwith another moiety, e.g., a non-carbohydrate (preferably cyclic)carrier to which is attached a carbohydrate ligand. A ribonucleotidesubunit in which the ribose sugar of the subunit has been so replaced isreferred to herein as a ribose replacement modification subunit (RRMS).A cyclic carrier may be a carbocyclic ring system, i.e., all ring atomsare carbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The carriers further include (i) at least one “backbone attachmentpoint”, preferably two “backbone attachment points” and (ii) at leastone “tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

In one aspect, the invention features, a compound having the structureshown in formula (CI)

A and B are independently for each occurrence hydrogen, protectinggroup, optionally substituted aliphatic, optionally substituted aryl,optionally substituted heteroaryl, polyethyleneglycol (PEG), aphosphate, a diphosphate, a triphosphate, a phosphonate, aphosphonothioate, a phosphonodithioate, a phosphorothioate, aphosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, aphosphodiester, a phosphotriester, an activated phosphate group, anactivated phosphite group, a phosphoramidite, a solid support,—P(Z¹)(Z²)—O-nucleoside, or —P(Z¹)(Z²)—O-oligonucleotide; wherein Z¹ andZ² are each independently for each occurrence O, S, N(alkyl) oroptionally substituted alkyl;

J₁ and J₂ are independently O, S, NR^(N), optionally substituted alkyl,OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OC(O)O, NHC(O)NH,NHC(S)NH, OC(S)NH, OP(N(R^(P))₂)O, or OP(N(R^(P))₂); and

is cyclic group or acyclic group; preferably, the cyclic group isselected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl,quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably,the acyclic group is selected from serinol backbone or diethanolaminebackbone.

In preferred embodiments, ligand is a carbohydrate e.g. monosaccharide,disaccharide, trisaccharide, tetrasaccharide, polysaccharide.

In one embodiment, the compound is a pyrroline ring system as shown informula (CII)

wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, orSO₂NH;

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each independently foreach occurrence H, —CH₂OR^(a), or OR^(b),

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(O-linker-R^(L))—O-nucleoside, or—P(Z¹)(O-linker-R^(L))—O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, —R^(L), -linker-R^(L) orR³¹;

Z¹ is independently for each occurrence O or S;

Z² is independently for each occurrence O, S, N(alkyl) or optionallysubstituted alkyl; and

h is independently for each occurrence 1-20.

For the pyrroline-based click-carriers, R¹¹ is —CH₂OR^(a) and R³ isOR^(b); or R¹¹ is —CH₂OR^(a) and R⁹ is OR^(b); or R¹¹ is —CH₂OR^(a) andR¹⁷ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹ is OR^(b); or R¹³ is—CH₂OR^(a) and R¹⁵ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹⁷ is OR^(b). Incertain embodiments, CH₂OR^(a) and OR^(b) may be geminally substituted.For the 4-hydroxyproline-based carriers, R¹¹ is —CH₂OR^(a) and R¹⁷ isOR^(b). The pyrroline- and 4-hydroxyproline-based compounds maytherefore contain linkages (e.g., carbon-carbon bonds) wherein bondrotation is restricted about that particular linkage, e.g. restrictionresulting from the presence of a ring. Thus, CH₂OR^(a) and OR^(b) may becis or trans with respect to one another in any of the pairingsdelineated above Accordingly, all cis/trans isomers are expresslyincluded. The compounds may also contain one or more asymmetric centersand thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the compounds are expressly included (e.g., the centers bearingCH₂OR^(a) and OR^(b) can both have the R configuration; or both have theS configuration; or one center can have the R configuration and theother center can have the S configuration and vice versa).

In one embodiment, R¹¹ is CH₂OR^(a) and R⁹ is OR^(b).

In one embodiment, R^(b) is a solid support.

In one embodiment, carrier of formula (CII) is a phosphoramidite, i.e.,one of R^(a) or R^(b) is —P(O-alkyl)N(alkyl)₂, e.g.,—P(OCH₂CH₂CN)N(i-propyl)₂. In one embodiment, R^(b) is—P(O-alkyl)N(alkyl)₂.

In embodiment, the compound is a ribose ring system as shown in formula(CIII).

wherein:

X is O, S, NR^(N) or CR^(P) ₂;

B is independently for each occurrence hydrogen, optionally substitutednatural or non-natural nucleobase, optionally substituted naturalnucleobase conjugated with -linker-R^(L) or optionally substitutednon-natural nucleobase conjugated with -linker-R^(L);

R¹, R², R³, R⁴ and R⁵ are each independently for each occurrence H, OR⁶,F, N(R^(N))₂, or -J-linker-R_(L);

J is absent, O, S, NR^(N), OC(O)NH, NHC(O)O, C(O)NH, NHC(O), NHSO,NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH,OP(N(R^(P))₂)O, or OP(N(R^(P))₂);

R⁶ is independently for each occurrence hydrogen, hydroxyl protectinggroup, optionally substituted alkyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted alkenyl, optionally substituted heteroaryl,polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, aphosphonate, a phosphonothioate, a phosphonodithioate, aphosphorothioate, a phosphorothiolate, a phosphorodithioate, aphosphorothiolothionate, a phosphodiester, a phosphotriester, anactivated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (CIII),—P(Z¹)(O-linker-R^(L))—O-nucleoside,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide, or—P(Z¹)(O-linker-R^(L))—O-formula (CIII);

R^(N) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl, optionally substituted heteroaryl or anamino protecting group;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl oroptionally substituted heteroaryl;

R^(L) is hydrogen or a ligand;

Z¹ and Z² are each independently for each occurrence O, S N(alkyl) oroptionally substituted alkyl; and

provided that R^(L) is present at least once and further provided thatR^(L) is a ligand at least once.

In one embodiment, the carrier of formula (CI) is an acyclic group andis termed an “acyclic carrier”. Preferred acyclic carriers can have thestructure shown in formula (CIV) or formula (CV) below.

In one embodiment, the compound is an acyclic carrier having thestructure shown in formula (CIV).

wherein:

W is absent, O, S and N(R^(N)), where R^(N) is independently for eachoccurrence H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted heteroaryl or an amino protecting group;

E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, or SO₂NH;

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(O-linker-R^(L))—O-nucleoside, or—P(Z¹)(O-linker-R^(L))—O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, —R^(L), -linker-R^(L) orR³¹;

Z¹ is independently for each occurrence O or S;

Z² is independently for each occurrence O, S, N(alkyl) or optionallysubstituted alkyl;

h is independently for each occurrence 1-20; and

r, s and t are each independently for each occurrence 0, 1, 2 or 3.

When r and s are different, then the tertiary carbon can be either the Ror S configuration. In preferred embodiments, x and y are one and z iszero (e.g. carrier is based on serinol). The acyclic carriers canoptionally be substituted, e.g. with hydroxy, alkoxy, perhaloalky.

In one embodiment, the compound is an acyclic carrier having thestructure shown in formula (CV)

wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, orSO₂NH;

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (I),—P(Z¹)(O-linker-R^(L))—O-nucleoside, or—P(Z¹)(O-linker-R^(L))—O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, —R^(L), -linker-R^(L) orR³¹;

Z¹ is independently for each occurrence O or S;

Z² is independently for each occurrence O, S, N(alkyl) or optionallysubstituted alkyl; and

h is independently for each occurrence 1-20; and

r and s are each independently for each occurrence 0, 1, 2 or 3. Inaddition to the cyclic carriers described herein, RRMS can includecyclic and acyclic carriers described in copending and co-owned U.S.application Ser. No. 10/916,185 filed Aug. 10, 2004, U.S. applicationSer. No. 10/946,873 filed Sep. 21, 2004, and U.S. application Ser. No.10/985,426, filed Nov. 9, 2004, U.S. application Ser. No. 10/833,934,filed Aug. 3, 2007 U.S. application Ser. No. 11/115,989 filed Apr. 27,2005, and U.S. application Ser. No. 11/119,533, filed Apr. 29, 2005,contents of each are hereby incorporated by reference for all purposes.

Accordingly, in one aspect, the invention features, a monomer having thestructure shown in formula (I)

wherein:

A and B are each independently for each occurrence O, N(R^(N)) or S;

R^(N) is independently for each occurrence H or C₁-C₆ alkyl;

X and Y are each independently for each occurrence H, a protectinggroup, a phosphate group, a phosphodiester group, an activated phosphategroup, an activated phosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a polymer, a nucleotide, a nucleoside,—P(Z′)(Z″)O-Linker-OP(Z′″)(Z″″)O-oligonucleotide, an oligonucleotide,—P(Z′)(Z″)-formula(I), —P(Z′)(Z″)— or -Linker-R;

R is L^(G) or has the structure shown below:

L^(G) is independently for each occurrence a ligand, e.g., carbohydrate,e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide,polysaccharide; and

Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S.

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR⁸, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R⁸), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R⁸ is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between 1-24atoms, preferably 4-24 atoms, preferably 6-18 atoms, more preferably8-18 atoms, and most preferably 8-16 atoms.

In one embodiment, the linker is—[(P-Q″-R)_(q)—X—(P′-Q′″-R′)_(q′)]_(q″)-T-, wherein:

P, R, T, P′, R′ and T are each independently for each occurrence absent,CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, CH═N—O,

or heterocyclyl;

Q″ and Q′″ are each independently for each occurrence absent,—(CH₂)_(n)—, —C(R¹)(R²)(CH₂)_(n)—, —(CH₂)_(n)C(R¹)(R²)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is absent or a cleavable linking group;

R^(a) is H or an amino acid side chain;

R¹ and R² are each independently for each occurrence H, CH₃, OH, SH orN(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

-   -   q, q′ and q″ are each independently for each occurrence 0-20 and        wherein the repeating unit can be the same or different;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup.

In certain embodiments, the linker is a branched linker. The branchpointof the branched linker may be at least trivalent, but may be atetravalent, pentavalent or hexavalent atom, or a group presenting suchmultiple valencies. In certain embodiments, the branchpoint is, —N,—N(Q)-C, —O—C, —S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or—N(Q)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above. As used herein,“carbohydrate” refers to a compound which is either a carbohydrate perse made up of one or more monosaccharide units having at least 6 carbonatoms (which may be linear, branched or cyclic) with an oxygen, nitrogenor sulfur atom bonded to each carbon atom; or a compound having as apart thereof a carbohydrate moiety made up of one or more monosaccharideunits each having at least six carbon atoms (which may be linear,branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded toeach carbon atom. Representative carbohydrates include the sugars(mono-, di-, tri- and oligosaccharides containing from about 4-9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C₅and above (preferably C₅-C₈) sugars; di- and trisaccharides includesugars having two or three monosaccharide units (preferably C₅-C₈).

The term “monosaccharide” embraces radicals of allose, altrose,arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol,L-fucitol, fucosamine, fucose, fuculose, galactosamine,D-galactosaminitol, N-acetyl-galactosamine, galactose, glucosamine,N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate,gulose glyceraldehyde, L-glycero-D-mannos-heptose, glycerol, glycerone,gulose, idose, lyxose, mannosamine, nannose, mannose-6-phosphate,psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose,ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaricacid, threose, xylose and xylulose. The monosaccharide can be in D- orL-configuration. The monosaccharide may further be a deoxy sugar(alcoholic hydroxy group replaced by hydrogen), amino sugar (alcoholichydroxy group replaced by amino group), a thio sugar (alcoholic hydroxygroup replaced by thiol, or C═O replaced by C═S, or a ring oxygen ofcyclic form replaced by sulfur), a seleno sugar, a telluro sugar, an azasugar (ring carbon replaced by nitrogen), an imino sugar (ring oxygenreplaced by nitrogen), a phosphano sugar (ring oxygen replaced withphosphorus), a phospha sugar (ring carbon replaced with phosphorus), aC-substituted monosaccharide (hydrogen at a non-terminal carbon atomreplaced with carbon), an unsaturated monosaccharide, an alditol(carbonyl group replaced with CHOH group), aldonic acid (aldehydic groupreplaced by carboxy group), a ketoaldonic acid, a uronic acid, analdaric acid, and so forth. Amino sugars include amino monosaccharides,preferably galactosamine, glucosamine, mannosamine, fucosamine,quinovosamine, neuraminic acid, muramic acid, lactosediamine, acosamine,bacillosamine, daunosamine, desosamine, forosamine, garosamine,kanosamine, kansosamine, mycaminose, mycosamine, perosamine,pneumosamine, purpurosamine, rhodosamine. It is understood that themonosaccharide and the like can be further substituted.

The terms “disaccharide”, “trisaccharide” and “polysaccharide” embraceradicals of abequose, acrabose, amicetose, amylopectin, amylose, apiose,arcanose, ascarylose, ascorbic acid, boivinose, cellobiose, celotriose,cellulose, chacotriose, chaicose, chitin, colitose, cyclodextrin,cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, diginose, digitalose,digitoxose, evalose, evermirose, fiuctooligosachharide,galto-oligosaccharide, gentianose, gentiobiose, glucan, glucogen,glycogen, hamamelose, heparin, inulin, isolevogiucosenone, isornaltose,isornaltotriose, isopanose, kojibiose, lactose, lactosamnine,lactosediamine, laminarabiose, levoglucosan, levoglucosenone, β-maltose,maltriose, mannan-oligosaccharide, manninotriose, melezitose, melibiose,muramic acid, mycarose, mycinose, neuraminic acid, nigerose,nojirimycin, noviose, oleandrose, panose, paratose, planteose,primeverose, raffinose, rhodinose, rutinose, sarmentose, sedoheptulose,sedoheptulosan, solatriose, sophorose, stachyose, streptose, sucrose,α,α-trehalose, tiehalosamine, turanose, tyvelose, xylobiose,umbelliferose and the like. Further, it is understood that the“disaccharide”, “trisaccharide” and “polysaccharide” and the like can befurther substituted. Disaccharide also includes amino sugars and theirderivatives, particularly, a mycaminose derivatized at the C-4′ positionor a 4 deoxy-3-amino-glucose derivatized at the C-6′ position.

In one embodiment, the compound having the structure shown in formula(I′):

wherein:

A and B are each independently for each occurrence O, N(R^(N)) or S;

X and Y are each independently for each occurrence H, a protectinggroup, a phosphate group, a phosphodiester group, an activated phosphategroup, an activated phosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a polymer, a nucleotide, a nucleoside,—P(Z′)(Z″)O—R¹-Q′-R²—OP(Z′″)(Z″″)O-oligonucleotide, or anoligonucleotide, —P(Z′)(Z″)-formula(I), —P(Z′)(Z″)— or -Q-R;

R is L¹ or has the structure shown in formula (II)-(V):

q^(2A), q^(2B), q^(3A), q^(3B), q^(4A), q^(4B), q^(5A), q^(5B) andq^(5C) represent independently for each occurrence 0-20 and wherein therepeating unit can be the same or different;

Q and Q′ are independently for each occurrence is absent,—(P⁷-Q⁷-R⁷)_(p)-T⁷- or -T⁷-Q⁷-T^(7′)-B-T^(8′)-Q₈-T⁸;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),P⁷, T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B),T^(5C), T⁷, T^(7′), T⁸ and T^(8′) are each independently for eachoccurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

B is —CH₂—N(B^(L))—CH₂—;

B^(L) is -T^(B)-Q^(B)-T^(B′)-R^(x);

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C),Q⁷, Q⁸ and Q^(B) are independently for each occurrence absent, alkylene,substituted alkylene and wherein one or more methylenes can beinterrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)),C(R′)═C(R′), C≡C or C(O);

T^(B) and T^(B′) are each independently for each occurrence absent, CO,NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH₂, CH₂NH or CH₂O;

R^(x) is a lipophile (e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide, polysaccharide), an endosomolytic component, a steroid(e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid;

R¹, R², R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B),R^(5C), R⁷ are each independently for each occurrence absent, NH, O, S,CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L¹, L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) are each independently for each occurrence a carbohydrate, e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide and polysaccharide;

R′ and R″ are each independently H, C¹-C₆ alkyl, OH, SH, or N(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

R^(a) is H or amino acid side chain;

Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S; prepresent independently for each occurrence 0-20.

In some embodiments, the formula (I′) has the structure

In some embodiments, the formula (I′) has the structure

In some embodiments, the formula (I′) has the structure

In some embodiments, the formula (I′) has the structure

In some embodiments, the formula (I′) has the structure

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some preferred embodiments, R is

In some preferred embodiments, R is

In some preferred embodiments, formula (I) has the structure

In some embodiments R is

In some embodiments monomer of formula (I) has the structure

In some embodiments monomer of formula (I) has the structure

In some embodiments monomer of formula (I) has the structure

In some embodiments monomer of formula (I) has the structure

In some embodiments monomer of formula (I) has the structure

In some embodiments monomer of formula (I) has the structure

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some embodiments, R is

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments both L^(2A) and L^(2B) are the same.

In some embodiments both L^(2A) and L^(2B) are different.

In some preferred embodiments both L^(3A) and L^(3B) are the same.

In some embodiments both L^(3A) and L^(3B) are different.

In some preferred embodiments both L^(4A) and L^(4B) are the same.

In some embodiments both L^(4A) and L^(4B) are different.

In some preferred embodiments all of L^(5A), L^(5B) and L^(5C) are thesame.

In some embodiments two of L^(5A), L^(5B) and L^(5C) are the same.

In some embodiments L^(5A) and L^(5B) are the same.

In some embodiments L^(5A) and L^(5C) are the same.

In some embodiments L^(5B) and L^(5C) are the same.

In another aspect, the invention features, an iRNA agent comprising atleast one monomer of formula (I).

In some embodiments, the iRNA agent will comprise 1, 2, 3, 4 or 5monomers of formula (I), more preferably 1, 2 or 3 monomers of formula(I), more preferably 1 or 2 monomers of formula (I), even morepreferably only one monomer of formula (I).

In some embodiments, all the monomers of formula (I) are on the samestrand of a double stranded iRNA agent.

In some embodiments, the monomers of formula (I) are on the separatestrands of a double strand of an iRNA agent.

In some embodiments, all monomers of formula (I) in an iRNA agent arethe same.

In some embodiments, the monomers of formula (I) in an iRNA agent areall different.

In some embodiments, only some monomers of formula (I) in an iRNA agentare the same.

In some embodiments, the monomers of formula (I) will be next to eachother in the iRNA agent.

In some embodiments, the monomers of formula (I) will not be next toeach other in the iRNA agent.

In some embodiments, the monomer of formula (I) will be on the 5′-end,3′-end, at an internal position, both the 3′- and the 5′-end, both5′-end and an internal position, both 3′-end and internal position, andat all three positions (5′-end, 3′-end and an internal position) of theiRNA agent.

In some preferred embodiments, R^(x) is cholesterol.

In some preferred embodiments, R^(x) is lithocholic.

In some preferred embodiments, R^(x) is oleyl lithocholic.

In some preferred embodiments, R^(x) has the structure

In some preferred embodiments, B^(L) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

wherein Y is O or S and n is 3-6.

In some preferred embodiments, formula (I) has the structure

wherein Y is O or S and n is 3-6.

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

wherein X is O or S.

In some preferred embodiments, formula (I) has the structure

wherein R is OH or NHCOOH.

In some preferred embodiments, formula (I) has the structure

wherein R is OH or NHCOOH.

In some preferred embodiments, monomer of formula (I) is linked to theiRNA agent through a linker of formula (VII)

wherein R is O or S.

In some preferred embodiments, formula (I) has the structure

wherein R is OH or NHCOOH.

In some preferred embodiments, formula (I) has the structure

In some preferred embodiments, formula (I) has the structure

where in R is OH or NHCOOH.

In some preferred embodiments, formula (I) has the structure

wherein R is OH or NHCOOH.

In some preferred embodiments, formula (I) has the structure

wherein R is OH or NHCOOH.

In some preferred embodiments, formula (I) has the structure

wherein R is OH or NHCOOH.

In some embodiments, the iRNA agent will have a monomer with thestructure shown in formula (VI) in addition to monomer of formula (I)

wherein X⁶ and Y⁶ are each independently H, OH, a hydroxyl protectinggroup, a phosphate group, a phosphodiester group, an activated phosphategroup, an activated phosphite group, a phosphoramidite, a solid support,—P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, asteroid, a polymer, —P(Z′)(Z″)O—R¹-Q′-R²—OP(Z′″)(Z″″)O-oligonucleotide,a nucleotide, or an oligonucleotide, —P(Z′)(Z″)-formula(I) or—P(Z′)(Z″)—;

Q⁶ is absent or —(P⁶-Q⁶-R⁶)_(v)-T⁶-;

P⁶ and T⁶ are each independently for each occurrence absent, CO, NH, O,S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q⁶ is independently for each occurrence absent, substituted alkylenewherein one or more methylenes can be interepted or terminated by one ormore of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R′), C≡C or C(O);

R⁶ is independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

R′ and R″ are each independently H, C₁-C₆ alkyl OH, SH, N(R^(N))₂;

R^(N) is independently for each occurrence methyl, ethyl, propyl,isopropyl, butyl or benzyl;

R^(a) is H or amino acid side chain;

Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S;

v represent independently for each occurrence 0-20;

R^(L) is a lipophile (e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide,disaccharide, trisaccharide, tetrasaccharide, oligosaccharide,polysaccharide), an endosomolytic component, a steroid (e.g., uvaol,hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid.

In some embodiments, one or more, e.g., 1, 2, 3, 4 or 5, monomers offormula (VI) in addition to one or more, e.g. 1, 2, 3, 4, or 5, monomersof formula (I) are present in the iRNA agent.

In some preferred embodiments only 1 monomer of formula (I) and 1monomer of formula (VI) are present in the iRNA agent.

In some embodiments, R^(L) is cholesterol.

In some embodiments, R^(L) is lithocholic.

In some embodiments, R^(L) is oleyl lithocholic.

In some embodiments, monomer of formula (I) is covalently linked withthe monomer of formula (VI).

In some preferred embodiments, monomer of formula (I) is linked with themonomer of formula (VI) through a phosphate linkage, e.g. aphosphodiester linkage, a phosphorothioate linkage, a phosphorodithioatelinkage.

In some preferred embodiments, monomer of formula (I) is linked to theiRNA agent through the monomer of formula (VI).

In some embodiments, monomer of formula (I) intervenes between the iRNAagent and the monomer of formula (VI).

In some embodiments, monomer of formula (I) and monomer of formula (II)are directly linked to each other.

In some embodiments, monomer of formula (I) and monomer of formula (II)are not directly linked to each other.

In some embodiments, monomer of formula (I) and monomer of formula (VI)are on separate strands of a double stranded iRNA agent.

In some embodiments, monomer of formula (I) and monomer of formula (VI)are on opposite terminal ends of the iRNA agent.

In some embodiments, monomer of formula (I) and monomer of formula (VI)are on the same terminal end of the iRNA agent.

In some embodiments, one of monomer of formula (I) or monomer of formula(VI) is at an internal position while the other is at a terminalposition of an iRNA agent.

In some embodiments, monomer of formula (I) and monomer of formula (VI)are both at an internal position of the iRNA agent.

In some preferred embodiments, monomer of formula (VI) has the structure

In some embodiments, the iRNA agent of the invention is selected fromthe group consisting of:

wherein the ligand is a PK modulator: X═O or S; Y═O or S; PEG stands forω-OH, ω-amino, ω-methoxy, ω-SH, ω-propargyl, ω-azido and ω-ligand PEGSwith MW between 200 and 100,000.

Endosomolytic Components

For macromolecular drugs and hydrophilic drug molecules, which cannoteasily cross bilayer membranes, entrapment in endosomal/lysosomalcompartments of the cell is thought to be the biggest hurdle foreffective delivery to their site of action. In recent years, a number ofapproaches and strategies have been devised to address this problem. Forliposomal formulations, the use of fusogenic lipids in the formulationhave been the most common approach (Singh, R. S., Goncalves, C. et al.(2004). On the Gene Delivery Efficacies of pH-Sensitive Cationic Lipidsvia Endosomal Protonation. A Chemical Biology Investigation. Chem. Biol.11, 713-723). Other components, which exhibit pH-sensitive endosomolyticactivity through protonation and/or pH-induced conformational changes,include charged polymers and peptides. Examples may be found in Hoffman,A. S., Stayton, P. S. et al. (2002). Design of “smart” polymers that candirect intracellular drug delivery. Polymers Adv. Technol. 13, 992-999;Kakudo, Chaki, T., S. et al. (2004). Transferrin-Modified LiposomesEquipped with a pH-Sensitive Fusogenic Peptide: An Artificial Viral-likeDelivery System. Biochemistry 436, 5618-5628; Yessine, M. A. and Leroux,J. C. (2004). Membrane-destabilizing polyanions: interaction with lipidbilayers and endosomal escape of biomacromolecules. Adv. Drug Deliv.Rev. 56, 999-1021; Oliveira, S., van Rooy, I. et al. (2007). Fusogenicpeptides enhance endosomal escape improving siRNA-induced silencing ofoncogenes. Int. J. Pharm. 331, 211-4. They have generally been used inthe context of drug delivery systems, such as liposomes or lipoplexes.For folate receptor-mediated delivery using liposomal formulations, forinstance, a pH-sensitive fusogenic peptide has been incorporated intothe liposomes and shown to enhance the activity through improving theunloading of drug during the uptake process (Turk, M. J., Reddy, J. A.et al. (2002). Characterization of a novel pH-sensitive peptide thatenhances drug release from folate-targeted liposomes at endosomal pHs.Biochim. Biophys. Acta 1559, 56-68).

In certain embodiments, the endosomolytic components of the presentinvention may be polyanionic peptides or peptidomimetics which showpH-dependent membrane activity and/or fusogenicity. A peptidomimetic maybe a small protein-like chain designed to mimic a peptide. Apeptidomimetic may arise from modification of an existing peptide inorder to alter the molecule's properties, or the synthesis of apeptide-like molecule using unnatural amino acids or their analogs. Incertain embodiments, they have improved stability and/or biologicalactivity when compared to a peptide. In certain embodiments, theendosomolytic component assumes its active conformation at endosomal pH(e.g., pH 5-6). The “active” conformation is that conformation in whichthe endosomolytic component promotes lysis of the endosome and/ortransport of the modular composition of the invention, or its any of itscomponents (e.g., a nucleic acid), from the endosome to the cytoplasm ofthe cell.

Libraries of compounds may be screened for their differential membraneactivity at endosomal pH versus neutral pH using a hemolysis assay.Promising candidates isolated by this method may be used as componentsof the modular compositions of the invention. A method for identifyingan endosomolytic component for use in the compositions and methods ofthe present invention may comprise: providing a library of compounds;contacting blood cells with the members of the library, wherein the pHof the medium in which the contact occurs is controlled; determiningwhether the compounds induce differential lysis of blood cells at a lowpH (e.g., about pH 5-6) versus neutral pH (e.g., about pH 7-8).

Exemplary endosomolytic components include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (SEQ ID NO:1)(Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and theirderivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). Incertain embodiments, the endosomolytic component may contain a chemicalgroup (e.g., an amino acid) which will undergo a change in charge orprotonation in response to a change in pH. The endosomolytic componentmay be linear or branched. Exemplary primary sequences of endosomolyticcomponents include H₂N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO₂H (SEQ ID NO:2); H₂N-(AALAEALAEALAEALAEALAEALAAAAGGC)-CO₂H (SEQ ID NO: 3); andH₂N-(ALEALAEALEALAEA)-CONH₂ (SEQ ID NO: 4).

In certain embodiments, more than one endosomolytic component may beincorporated into the iRNA agent of the invention. In some embodiments,this will entail incorporating more than one of the same endosomolyticcomponent into the iRNA agent in addition to the monomers of formula(I). In other embodiments, this will entail incorporating two or moredifferent endosomolytic components into iRNA agent in addition to themonomers of formula (I).

These endosomolytic components may mediate endosomal escape by, forexample, changing conformation at endosomal pH. In certain embodiments,the endosomolytic components may exist in a random coil conformation atneutral pH and rearrange to an amphipathic helix at endosomal pH. As aconsequence of this conformational transition, these peptides may insertinto the lipid membrane of the endosome, causing leakage of theendosomal contents into the cytoplasm. Because the conformationaltransition is pH-dependent, the endosomolytic components can displaylittle or no fusogenic activity while circulating in the blood (pH˜7.4).Fusogenic activity is defined as that activity which results indisruption of a lipid membrane by the endosomolytic component. Oneexample of fusogenic activity is the disruption of the endosomalmembrane by the endosomolytic component, leading to endosomal lysis orleakage and transport of one or more components of the modularcomposition of the invention (e.g., the nucleic acid) from the endosomeinto the cytoplasm.

In addition to the hemolysis assay described herein, suitableendosomolytic components can be tested and identified by a skilledartisan using other methods. For example, the ability of a compound torespond to, e.g., change charge depending on, the pH environment can betested by routine methods, e.g., in a cellular assay. In certainembodiments, a test compound is combined with or contacted with a cell,and the cell is allowed to internalize the test compound, e.g., byendocytosis. An endosome preparation can then be made from the contactedcells and the endosome preparation compared to an endosome preparationfrom control cells. A change, e.g., a decrease, in the endosome fractionfrom the contacted cell vs. the control cell indicates that the testcompound can function as a fusogenic agent. Alternatively, the contactedcell and control cell can be evaluated, e.g., by microscopy, e.g., bylight or electron microscopy, to determine a difference in the endosomepopulation in the cells. The test compound and/or the endosomes canlabeled, e.g., to quantify endosomal leakage.

In another type of assay, an iRNA agent described herein is constructedusing one or more test or putative fusogenic agents. The iRNA agent canbe labeled for easy visualization. The ability of the endosomolyticcomponent to promote endosomal escape, once the iRNA agent is taken upby the cell, can be evaluated, e.g., by preparation of an endosomepreparation, or by microscopy techniques, which enable visualization ofthe labeled iRNA agent in the cytoplasm of the cell. In certain otherembodiments, the inhibition of gene expression, or any otherphysiological parameter, may be used as a surrogate marker for endosomalescape.

In other embodiments, circular dichroism spectroscopy can be used toidentify compounds that exhibit a pH-dependent structural transition.

A two-step assay can also be performed, wherein a first assay evaluatesthe ability of a test compound alone to respond to changes in pH, and asecond assay evaluates the ability of a modular composition thatincludes the test compound to respond to changes in pH.

Peptides

Peptides suitable for use with the present invention can be a naturalpeptide, e.g. tat or antennopedia peptide, a synthetic peptide or apeptidomimetic. Furthermore, the peptide can be a modified peptide, forexample peptide can comprise non-peptide or pseudo-peptide linkages, andD-amino acids. A peptidomimetic (also referred to herein as anoligopeptidomimetic) is a molecule capable of folding into a definedthree-dimensional structure similar to a natural peptide. The attachmentof peptide and peptidomimetics to the oligonucleotide can affectpharmacokinetic distribution of the oligonucleotide, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, forexample).

TABLE 1 Exemplary Cell Permeation Peptides Cell Permeation SEQ IDPeptide NO: Amino acid Sequence Reference Penetratin 5 RQIKIWFQNRRMKWKKDerossi et al., J. Biol. Chem. 269: 10444, 1994 Tat fragment 6GRKKRRQRRRPPQC Vives et al., J. Biol. (48-60) Chem., 272: 16010, 1997Signal 7 GALFLGWLGAAGSTMGAWSQP Chaloin et al., Biochem. Sequence-basedKKKRKV Biophys. Res. peptide Commun., 243: 601, 1998 PVEC 8LLIILRRRIRKQAHAHSK Elmquist et al., Exp. Cell Res., 269: 237, 2001Transportan 9 GWTLNSAGYLLKINLKALAALA Pooga et al., FASEB J., KKIL12: 67, 1998 Amphiphilic 10 KLALKLALKALKAALKLA Oehlke et al., Mol.model peptide Ther., 2: 339, 2000 Arg₉ 11 RRRRRRRRRMitchell et al., J. Pept. Res., 56: 318, 2000 Bacterial cell 12KFFKFFKFFK wall permeating LL-37 13 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Cecropin P1 14 SWLSKTAKKLENSAKKRISEGIA IAIQGGPR α-defensin15 ACYCRIPACIAGERRYGTCIYQG RLWAFCC b-defensin 16 DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK Bactenecin 17 RKCRIVVIRVCR PR-39 18RRRPRPPYLPRPRPPPFFPPRLPPR IPPGFPPRFPPRFPGKR-NH2 Indolicidin 19ILPWKWPWWPWRR-NH2

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 20). A RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 21)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 6)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 5))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered tothe lipid is a cell targeting peptide such as anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingto tumors of a variety of other tissues, including the lung, kidney,spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001).Preferably, the RGD peptide will facilitate targeting of the lipidparticle to the kidney. The RGD peptide can be linear or cyclic, and canbe modified, e.g., glycosylated or methylated to facilitate targeting tospecific tissues. For example, a glycosylated RGD peptide can target atumor cell expressing αvβ₃ (Haubner et al., Jour. Nucl. Med.,42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an I_(v)θ₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the I_(v)-θ₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

iRNA Agents

The iRNA agent should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the iRNA agent, or a fragment thereof, can mediate downregulationof the target gene. (For ease of exposition the term nucleotide orribonucleotide is sometimes used herein in reference to one or moremonomeric subunits of an RNA agent. It will be understood herein thatthe usage of the term “ribonucleotide” or “nucleotide”, herein can, inthe case of a modified RNA or nucleotide surrogate, also refer to amodified nucleotide, or surrogate replacement moiety at one or morepositions.) Thus, the iRNA agent is or includes a region which is atleast partially, and in some embodiments fully, complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of the targetRNA, e.g., mRNA. Complementarity, or degree of homology with the targetstrand, is most critical in the antisense strand. While perfectcomplementarity, particularly in the antisense strand, is often desiredsome embodiments can include, particularly in the antisense strand, oneor more, or for example, 6, 5, 4, 3, 2, or fewer mismatches (withrespect to the target RNA). The mismatches, particularly in theantisense strand, are most tolerated in the terminal regions and ifpresent may be in a terminal region or regions, e.g., within 6, 5, 4, or3 nucleotides of the 5′ and/or 3′ termini. The sense strand need only besufficiently complementary with the antisense strand to maintain theover all double stranded character of the molecule.

As discussed elsewhere herein, and in the material incorporated byreference in its entirety, an iRNA agent will often be modified orinclude nucleoside surrogates. Single stranded regions of an iRNA agentwill often be modified or include nucleoside surrogates, e.g., theunpaired region or regions of a hairpin structure, e.g., a region whichlinks two complementary regions, can have modifications or nucleosidesurrogates. Modification to stabilize one or more 3′- or 5′-termini ofan iRNA agent, e.g., against exonucleases, or to favor the antisensesiRNA agent to enter into RISC are also envisioned. Modifications caninclude C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyllinkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethyleneglycol, hexaethylene glycol), special biotin or fluorescein reagentsthat come as phosphoramidites and that have another DMT-protectedhydroxyl group, allowing multiple couplings during RNA synthesis.

iRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencingcomplex)); and, molecules which are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed siRNA agents or shorter iRNA agentsherein. “siRNA agent or shorter iRNA agent” as used herein, refers to aniRNA agent, e.g., a double stranded RNA agent or single strand agent,that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or acleavage product thereof, can down regulate a target gene, e.g., byinducing RNAi with respect to a target RNA, wherein the target maycomprise an endogenous or pathogen target RNA.

Each strand of an siRNA agent can be equal to or less than 30, 25, 24,23, 22, 21, or 20 nucleotides in length. The strand may be at least 19nucleotides in length. For example, each strand can be between 21 and 25nucleotides in length. siRNA agents may have a duplex region of 17, 18,19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or moreoverhangs, or one or two 3′ overhangs, of 2-3 nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an iRNA agent may have one or more of the followingproperties:

-   -   (1) it may be of the Formula VII, VIII, IX or X set out in the        RNA Agent section below;    -   (2) if single stranded it may have a 5′ modification which        includes one or more phosphate groups or one or more analogs of        a phosphate group;    -   (3) it may, despite modifications, even to a very large number,        or all of the nucleosides, have an antisense strand that can        present bases (or modified bases) in the proper three        dimensional framework so as to be able to form correct base        pairing and form a duplex structure with a homologous target RNA        which is sufficient to allow down regulation of the target,        e.g., by cleavage of the target RNA;    -   (4) it may, despite modifications, even to a very large number,        or all of the nucleosides, still have “RNA-like” properties,        i.e., it may possess the overall structural, chemical and        physical properties of an RNA molecule, even though not        exclusively, or even partly, of ribonucleotide-based content.        For example, an iRNA agent can contain, e.g., a sense and/or an        antisense strand in which all of the nucleotide sugars contain        e.g., 2′ fluoro in place of 2′ hydroxyl. This        deoxyribonucleotide-containing agent can still be expected to        exhibit RNA-like properties. While not wishing to be bound by        theory, the electronegative fluorine prefers an axial        orientation when attached to the C2′ position of ribose. This        spatial preference of fluorine can, in turn, force the sugars to        adopt a C_(3′)-endo pucker. This is the same puckering mode as        observed in RNA molecules and gives rise to the        RNA-characteristic A-family-type helix. Further, since fluorine        is a good hydrogen bond acceptor, it can participate in the same        hydrogen bonding interactions with water molecules that are        known to stabilize RNA structures. A modified moiety at the 2′        sugar position may be able to enter into H bonding which is more        characteristic of the OH moiety of a ribonucleotide than the H        moiety of a deoxyribonucleotide. Certain iRNA agents will:        exhibit a C_(3′)-endo pucker in all, or at least 50, 75, 80, 85,        90, or 95% of its sugars; exhibit a C_(3′)-endo pucker in a        sufficient amount of its sugars that it can give rise to a the        RNA-characteristic A-family-type helix; will have no more than        20, 10, 5, 4, 3, 2, or 1 sugar which is not a C_(3′)-endo pucker        structure. Regardless of the nature of the modification, and        even though the RNA agent can contain deoxynucleotides or        modified deoxynucleotides, particularly in overhang or other        single strand regions, it is certain DNA molecules, or any        molecule in which more than 50, 60, or 70% of the nucleotides in        the molecule, or more than 50, 60, or 70% of the nucleotides in        a duplexed region are deoxyribonucleotides, or modified        deoxyribonucleotides which are deoxy at the 2′ position, are        excluded from the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents may be antisense withregard to the target molecule. In certain embodiments single strand iRNAagents are 5′ phosphorylated or include a phosphoryl analog at the 5′prime terminus. 5′-phosphate modifications include those which arecompatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2 (O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated ornon-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2 (S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂ (O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g., 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2 (O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g., RP(OH)(O)—O-5′-, (OH)₂ (O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.,RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a double stranded iRNA.)

A single strand iRNA agent may be sufficiently long that it can enterthe RISC and participate in RISC mediated cleavage of a target mRNA. Asingle strand iRNA agent is at least 14, and in other embodiments atleast 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certainembodiments, it is less than 200, 100, or 60 nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17,18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex regionwill may be equal to or less than 200, 100, or 50, in length. In certainembodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23,and 19 to 21 nucleotides pairs in length. The hairpin may have a singlestrand overhang or terminal unpaired region, in some embodiments at the3′, and in certain embodiments on the antisense side of the hairpin. Insome embodiments, the overhangs are 2-3 nucleotides in length.

A “double stranded (ds) iRNA agent” as used herein, is an iRNA agentwhich includes more than one, and in some cases two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded iRNA agent may be equal to orat least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It may be equal to or less than 200, 100, or 50, nucleotides inlength. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength.

The sense strand of a double stranded iRNA agent may be equal to or atleast 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. Itmay be equal to or less than 200, 100, or 50, nucleotides in length.Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double strand portion of a double stranded iRNA agent may be equalto or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40,or 60 nucleotide pairs in length. It may be equal to or less than 200,100, or 50, nucleotides pairs in length. Ranges may be 15-30, 17 to 23,19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the ds iRNA agent is sufficiently large that it canbe cleaved by an endogenous molecule, e.g., by Dicer, to produce smallerds iRNA agents, e.g., siRNAs agents

It may be desirable to modify one or both of the antisense and sensestrands of a double strand iRNA agent. In some cases they will have thesame modification or the same class of modification but in other casesthe sense and antisense strand will have different modifications, e.g.,in some cases it is desirable to modify only the sense strand. It may bedesirable to modify only the sense strand, e.g., to inactivate it, e.g.,the sense strand can be modified in order to inactivate the sense strandand prevent formation of an active siRNA/protein or RISC. This can beaccomplished by a modification which prevents 5′-phosphorylation of thesense strand, e.g., by modification with a 5′-O-methyl ribonucleotide(see Nykiinen et al., (2001) ATP requirements and small interfering RNAstructure in the RNA interference pathway. Cell 107, 309-321.) Othermodifications which prevent phosphorylation can also be used, e.g.,simply substituting the 5′-OH by H rather than O-Me. Alternatively, alarge bulky group may be added to the 5′-phosphate turning it into aphosphodiester linkage, though this may be less desirable asphosphodiesterases can cleave such a linkage and release a functionalsiRNA 5′-end. Antisense strand modifications include 5′ phosphorylationas well as any of the other 5′ modifications discussed herein,particularly the 5′ modifications discussed above in the section onsingle stranded iRNA molecules.

The sense and antisense strands may be chosen such that the ds iRNAagent includes a single strand or unpaired region at one or both ends ofthe molecule. Thus, a ds iRNA agent may contain sense and antisensestrands, paired to contain an overhang, e.g., one or two 5′ or 3′overhangs, or a 3′ overhang of 2-3 nucleotides. Many embodiments willhave a 3′ overhang. Certain siRNA agents will have single-strandedoverhangs, in some embodiments 3′ overhangs, of 1 or 2 or 3 nucleotidesin length at each end. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. 5′ ends may be phosphorylated.

In some embodiments, the length for the duplexed region is between 15and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe siRNA agent range discussed above. siRNA agents can resemble inlength and structure the natural Dicer processed products from longdsiRNAs. Embodiments in which the two strands of the siRNA agent arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and a 3′ overhang are also within the invention.

The isolated iRNA agents described herein, including ds iRNA agents andsiRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., atranscript of a gene that encodes a protein. For convenience, such mRNAis also referred to herein as mRNA to be silenced. Such a gene is alsoreferred to as a target gene. In general, the RNA to be silenced is anendogenous gene or a pathogen gene. In addition, RNAs other than mRNA,e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an siRNA agent of 21 to 23nucleotides.

As used herein, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA, such that the iRNA agent silencesproduction of protein encoded by the target mRNA. In another embodiment,the iRNA agent is “exactly complementary” to a target RNA, e.g., thetarget RNA and the iRNA agent anneal, for example to form a hybrid madeexclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can includean internal region (e.g., of at least 10 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in some embodiments, the iRNAagent specifically discriminates a single-nucleotide difference. In thiscase, the iRNA agent only mediates RNAi if exact complementary is foundin the region (e.g., within 7 nucleotides of) the single-nucleotidedifference.

As used herein, the term “oligonucleotide” refers to a nucleic acidmolecule (RNA or DNA) for example of length less than 100, 200, 300, or400 nucleotides.

RNA agents discussed herein include unmodified RNA as well as RNA whichhave been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, for example as occur naturally in the human body. The art hasoften referred to rare or unusual, but naturally occurring, RNAs asmodified RNAs, see, e.g., Limbach et al., (1994) Summary: the modifiednucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare orunusual RNAs, often termed modified RNAs (apparently because the aretypically the result of a post transcriptionally modification) arewithin the term unmodified RNA, as used herein. Modified RNA refers to amolecule in which one or more of the components of the nucleic acid,namely sugars, bases, and phosphate moieties, are different from thatwhich occur in nature, for example, different from that which occurs inthe human body. While they are referred to as modified “RNAs,” they willof course, because of the modification, include molecules which are notRNAs. Nucleoside surrogates are molecules in which the ribophosphatebackbone is replaced with a non-ribophosphate construct that allows thebases to the presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of all of the above are discussed herein.

Much of the discussion below refers to single strand molecules. In manyembodiments of the invention a double stranded iRNA agent, e.g., apartially double stranded iRNA agent, is envisioned. Thus, it isunderstood that that double stranded structures (e.g., where twoseparate molecules are contacted to form the double stranded region orwhere the double stranded region is formed by intramolecular pairing(e.g., a hairpin structure)) made of the single stranded structuresdescribed below are within the invention. Lengths are describedelsewhere herein.

As nucleic acids are polymers of subunits, many of the modificationsdescribed below occur at a position which is repeated within a nucleicacid, e.g., a modification of a base, or a phosphate moiety, or the anon-linking O of a phosphate moiety. In some cases the modification willoccur at all of the subject positions in the nucleic acid but in manycases it will not. By way of example, a modification may only occur at a3′ or 5′ terminal position, may only occur in a terminal regions, e.g.,at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10nucleotides of a strand. A modification may occur in a double strandregion, a single strand region, or in both. A modification may occuronly in the double strand region of an RNA or may only occur in a singlestrand region of an RNA. E.g., a phosphorothioate modification at anon-linking O position may only occur at one or both termini, may onlyoccur in a terminal regions, e.g., at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, ormay occur in double strand and single strand regions, particularly attermini. The 5′ end or ends can be phosphorylated.

In some embodiments it is possible, e.g., to enhance stability, toinclude particular bases in overhangs, or to include modifiednucleotides or nucleotide surrogates, in single strand overhangs, e.g.,in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to includepurine nucleotides in overhangs. In some embodiments all or some of thebases in a 3′ or 5′ overhang will be modified, e.g., with a modificationdescribed herein. Modifications can include, e.g., the use ofmodifications at the 2′ OH group of the ribose sugar, e.g., the use ofdeoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides,and modifications in the phosphate group, e.g., phosphothioatemodifications. Overhangs need not be homologous with the targetsequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula VII represents a portion of aribonucleic acid. The basic components are the ribose sugar, the base,the terminal phosphates, and phosphate internucleotide linkers. Wherethe bases are naturally occurring bases, e.g., adenine, uracil, guanineor cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (asdepicted) and W, X, Y, and Z are all O, Formula VII represents anaturally occurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the above RNA components can confer improved properties, and,e.g., can render oligoribonucleotides more stable to nucleases.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

-   -   (i) alteration, e.g., replacement, of one or both of the        non-linking (X and Y) phosphate oxygens and/or of one or more of        the linking (W and Z) phosphate oxygens (When the phosphate is        in the terminal position, one of the positions W or Z will not        link the phosphate to an additional element in a naturally        occurring ribonucleic acid. However, for simplicity of        terminology, except where otherwise noted, the W position at the        5′ end of a nucleic acid and the terminal Z position at the 3′        end of a nucleic acid, are within the term “linking phosphate        oxygens” as used herein);    -   (ii) alteration, e.g., replacement, of a constituent of the        ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar;    -   (iii) wholesale replacement of the phosphate moiety (bracket I)        with “dephospho” linkers;    -   (iv) modification or replacement of a naturally occurring base;    -   (v) replacement or modification of the ribose-phosphate backbone        (bracket II);    -   (vi) modification of the 3′ end or 5′ end of the RNA, e.g.,        removal, modification or replacement of a terminal phosphate        group or conjugation of a moiety, e.g., a fluorescently labeled        moiety, to either the 3′ or 5′ end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule.

It is understood that the actual electronic structure of some chemicalentities cannot be adequately represented by only one canonical form(i.e., Lewis structure). While not wishing to be bound by theory, theactual structure can instead be some hybrid or weighted average of twoor more canonical forms, known collectively as resonance forms orstructures. Resonance structures are not discrete chemical entities andexist only on paper. They differ from one another only in the placementor “localization” of the bonding and nonbonding electrons for aparticular chemical entity. It can be possible for one resonancestructure to contribute to a greater extent to the hybrid than theothers. Thus, the written and graphical descriptions of the embodimentsof the present invention are made in terms of what the art recognizes asthe predominant resonance form for a particular species. For example,any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)would be represented by X═O and Y═N in the above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-linking oxygen atoms (i.e., X and Yin Formula 1 above).

However, the phosphate group can be modified by replacing one of theoxygens with a different substituent. One result of this modification toRNA phosphate backbones can be increased resistance of theoligoribonucleotide to nucleolytic breakdown. Thus while not wishing tobe bound by theory, it can be desirable in some embodiments to introducealterations which result in either an uncharged linker or a chargedlinker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. Unlike the situation where only one of X or Y isaltered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of oligoribonucleotides diastereomers.Diastereomer formation can result in a preparation in which theindividual diastereomers exhibit varying resistance to nucleases.Further, the hybridization affinity of RNA containing chiral phosphategroups can be lower relative to the corresponding unmodified RNAspecies. Thus, while not wishing to be bound by theory, modifications toboth X and Y which eliminate the chiral center, e.g., phosphorodithioateformation, may be desirable in that they cannot produce diastereomermixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Replacement of X and/or Y with sulfur is possible.

The phosphate linker can also be modified by replacement of a linkingoxygen (i.e., W or Z in Formula 1) with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylenephosphonates). The replacement can occur at a terminaloxygen (position W (3′) or position Z (5′). Replacement of W with carbonor Z with nitrogen is possible.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a 2′alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecularnucleophilic attack on the linker phosphorus atom. Again, while notwishing to be bound by theory, it can be desirable to some embodimentsto introduce alterations in which alkoxide formation at the 2′ positionis not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthythat oligonucleotides containing only the methoxyethyl group (MOE),(OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparableto those modified with the robust phosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e., deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g., NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; andalkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionallysubstituted with e.g., an amino functionality. Other substitutents ofcertain embodiments include 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

Modified RNA's can also include “abasic” sugars, which lack a nucleobaseat C-1′. These abasic sugars can also be further contain modificationsat one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors (cf. Bracket I in Formula 1 above). While not wishing to bebound by theory, it is believed that since the charged phosphodiestergroup is the reaction center in nucleolytic degradation, its replacementwith neutral structural mimics should impart enhanced nucleasestability. Again, while not wishing to be bound by theory, it can bedesirable, in some embodiment, to introduce alterations in which thecharged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.In certain embodiments, replacements may include themethylenecarbonylamino and methylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates (see Bracket II of Formula 1 above).While not wishing to be bound by theory, it is believed that the absenceof a repetitively charged backbone diminishes binding to proteins thatrecognize polyanions (e.g., nucleases). Again, while not wishing to bebound by theory, it can be desirable in some embodiment, to introducealterations in which the bases are tethered by a neutral surrogatebackbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNAsurrogates may be used.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)O—, —(CH₂)S—, O(CH₂CH₂O)CH₂CH₂OH (e.g., n=3 or 6),abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,disulfide, thiourea, sulfonamide, or morpholino, or biotin andfluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g., biotin), transport/absorption facilitators (e.g.,aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,imidazole, bisimidazole, histamine, imidazole clusters,acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogs. E.g., in certain embodiments iRNA agents, especially antisensestrands, are 5′ phosphorylated or include a phosphoryl analog at the 5′prime terminus. 5′-phosphate modifications include those which arecompatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2 (O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated ornon-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2 (S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2 (O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g., 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2 (O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g., RP(OH)(O)—O-5′-, (OH)₂ (O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.,RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for increasing resistance todegradation.

Terminal modifications can also be useful for monitoring distribution,and in such cases the groups to be added may include fluorophores, e.g.,fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications canalso be useful for enhancing uptake, useful modifications for thisinclude cholesterol. Terminal modifications can also be useful forcross-linking an RNA agent to another moiety; modifications useful forthis include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA.

These bases can be modified or replaced to provide RNA's having improvedproperties. E.g., nuclease resistant oligoribonucleotides can beprepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the above basesand “universal bases” can be employed. Examples include 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine,7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are not used for promoting stability, but theycan be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and2 amino purine, are fluorescent. Modified bases can reduce targetspecificity. This may be taken into consideration in the design of iRNAagents.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate RNAs

One can evaluate a candidate RNA agent, e.g., a modified RNA, for aselected property by exposing the agent or modified molecule and acontrol molecule to the appropriate conditions and evaluating for thepresence of the selected property. For example, resistance to adegradent can be evaluated as follows. A candidate modified RNA (and acontrol molecule, usually the unmodified form) can be exposed todegradative conditions, e.g., exposed to a milieu, which includes adegradative agent, e.g., a nuclease. E.g., one can use a biologicalsample, e.g., one that is similar to a milieu, which might beencountered, in therapeutic use, e.g., blood or a cellular fraction,e.g., a cell-free homogenate or disrupted cells. The candidate andcontrol could then be evaluated for resistance to degradation by any ofa number of approaches. For example, the candidate and control could belabeled prior to exposure, with, e.g., a radioactive or enzymatic label,or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA'scan be incubated with the degradative agent, and optionally a control,e.g., an inactivated, e.g., heat inactivated, degradative agent. Aphysical parameter, e.g., size, of the modified and control moleculesare then determined. They can be determined by a physical method, e.g.,by polyacrylamide gel electrophoresis or a sizing column, to assesswhether the molecule has maintained its original length, or assessedfunctionally. Alternatively, Northern blot analysis can be used to assaythe length of an unlabeled modified molecule.

A functional assay can also be used to evaluate the candidate agent. Afunctional assay can be applied initially or after an earliernon-functional assay, (e.g., assay for resistance to degradation) todetermine if the modification alters the ability of the molecule tosilence gene expression. For example, a cell, e.g., a mammalian cell,such as a mouse or human cell, can be co-transfected with a plasmidexpressing a fluorescent protein, e.g., GFP, and a candidate RNA agenthomologous to the transcript encoding the fluorescent protein (see,e.g., WO 00/44914). For example, a modified dsiRNA homologous to the GFPmRNA can be assayed for the ability to inhibit GFP expression bymonitoring for a decrease in cell fluorescence, as compared to a controlcell, in which the transfection did not include the candidate dsiRNA,e.g., controls with no agent added and/or controls with a non-modifiedRNA added. Efficacy of the candidate agent on gene expression can beassessed by comparing cell fluorescence in the presence of the modifiedand unmodified dsiRNA agents.

In an alternative functional assay, a candidate dsiRNA agent homologousto an endogenous mouse gene, for example, a maternally expressed gene,such as c-mos, can be injected into an immature mouse oocyte to assessthe ability of the agent to inhibit gene expression in vivo (see, e.g.,WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintainarrest in metaphase II, can be monitored as an indicator that the agentis inhibiting expression. For example, cleavage of c-mos mRNA by adsiRNA agent would cause the oocyte to exit metaphase arrest andinitiate parthenogenetic development (Colledge et al. Nature 370: 65-68,1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of themodified agent on target RNA levels can be verified by Northern blot toassay for a decrease in the level of target mRNA, or by Western blot toassay for a decrease in the level of target protein, as compared to anegative control. Controls can include cells in which with no agent isadded and/or cells in which a non-modified RNA is added.

RNA Structure References

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be with solid phase synthesis, see for example“Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRLPress, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed.F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis, Chapter 2,Oligoribonucleotide synthesis, Chapter 3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4,Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein. Modification described inWO 00/44895, WO01/75164, or WO02/44321 can be used herein.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7, 651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Base References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.Additional references can be disclosed in the above section on basemodifications.

Additional RNA Agents

Certain RNA agents have the following structure (Formula VIII):

wherein:

R¹, R², and R³ are independently H, (i.e., abasic nucleotides), adenine,guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine,nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl andother alkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 5-halouracil and cytosine,5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases;

R⁴, R⁵, and R⁶ are independently OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸; O(CH₂)R⁹;O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R)₂; NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹;NHC(O)R⁸; cyano; mercapto, SR⁸; alkyl-thio-alkyl; alkyl, aralkyl,cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may beoptionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl,alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl,aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl,alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl,acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ together combine with R⁷ toform an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′carbons;

A¹ is:

H; OH, OCH₃, W¹; an abasic nucleotide; or absent;

(in some embodiments, A1, especially with regard to anti-sense strands,is chosen from 5′-monophosphate ((HO)₂ (O)P—O-5′), 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated ornon-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-monothiophosphate (phosphorothioate; (HO)₂ (S)P—O-5′),5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂ (O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g., 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂ (O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g., RP(OH)(O)—O-5′-, (OH)₂ (O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.,RP(OH)(O)—O-5′-));

A² is:

A³ is:

A⁴ is:

H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent;

W¹ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰;O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰;O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰,O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰ or —O—;

W⁴ is O, CH₂, NH, or S;

X¹, X², X³, and X⁴ are each independently O or S;

Y¹, Y², Y³, and Y⁴ are each independently OH, O⁻, OR, S, Se, BH₃ ⁻, H,NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each ofwhich may be optionally substituted;

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S;

Z⁴ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰,O(CH₂)_(n)SS(CH₂)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰,O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰,O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰;

x is 5-100, chosen to comply with a length for an RNA agent describedherein;

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂—]covalently bound bridge between the sugar 2′ and 4′ carbons;

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar;

R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, diheteroaryl amino, or amino acid;

R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes);sulfur, silicon, boron or ester protecting group; intercalating agents(e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g., EDTA), lipohilic carriers (cholesterol, cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelledmarkers, enzymes, haptens (e.g., biotin), transport/absorptionfacilitators (e.g., aspirin, vitamin E, folic acid), syntheticribonucleases (e.g., imidazole, bisimidazole, histamine, imidazoleclusters, acridine-imidazole conjugates, Eu3+ complexes oftetraazamacrocycles); or an RNA agent;

m is 0-1,000,000;

n is 0-20.

Q is a spacer selected from the group consisting of abasic sugar, amide,carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea,sulfonamide, or morpholino, biotin or fluorescein reagents.

Certain RNA agents in which the entire phosphate group has been replacedhave the following structure (Formula IX):

wherein:

A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent, wherein

L is a linker, wherein one or both L may be present or absent and isselected from the group consisting of CH₂ (CH₂)_(g); N(CH₂)_(g);O(CH₂)_(g); S(CH₂)_(g);

G is a functional group selected from the group consisting of siloxane,carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxidelinker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino;R¹⁰, R²⁰, and R³⁰ are independently H, (i.e., abasic nucleotides),adenine, guanine, cytosine and uracil, inosine, thymine, xanthine,hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases;R⁴⁰, R⁵⁰, and R⁶⁰ are independently OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR;O(CH₂)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂R⁹; NHC(O)R⁸; cyano; mercapto, SR⁷;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridgebetween the sugar 2′ and 4′ carbons;x is 5-100 or chosen to comply with a length for an RNA agent describedherein;R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an[—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons;R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar;R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, diheteroaryl amino, or amino acid;m is 0-1,000,000;n is 0-20;g is 0-2.

Certain nucleoside surrogates have the following structure (Formula X):SLR¹⁰⁰-(M-SLR²⁰⁰)_(x)-M-SLR³⁰⁰   FORMULA X

wherein:

S is a nucleoside surrogate selected from the group consisting ofmophilino, cyclobutyl, pyrrolidine and peptide nucleic acid;

L is a linker and is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may beabsent;

M is an amide bond; sulfonamide; sulfinate; phosphate group; modifiedphosphate group as described herein; or may be absent;

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are independently H (i.e., abasic nucleotides),adenine, guanine, cytosine and uracil, inosine, thymine, xanthine,hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2, 4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases;

x is 5-100, or chosen to comply with a length for an RNA agent describedherein;

g is 0-2.

Definitions

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine. The term “alkyl” refers to saturated and unsaturatednon-aromatic hydrocarbon chains that may be a straight chain or branchedchain, containing the indicated number of carbon atoms (these includewithout limitation propyl, allyl, or propargyl), which may be optionallyinserted with N, O, or S. For example, C₁-C₁₀ indicates that the groupmay have from 1 to 10 (inclusive) carbon atoms in it. The term “alkoxy”refers to an —O-alkyl radical. The term “alkylene” refers to a divalentalkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent speciesof the structure —O—R—O—, in which R represents an alkylene. The term“aminoalkyl” refers to an alkyl substituted with an amino The term“mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an—S-alkyl radical.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include trizolyl, tetrazolyl, piperazinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to:

halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio,arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl,alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro,alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino,hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl,aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonicacid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, andaliphatic. It is understood that the substituent can be furthersubstituted.

Palindromes

The iRNA agents of the invention can target more than one RNA region.For example, an iRNA agent can include a first and second sequence thatare sufficiently complementary to each other to hybridize. The firstsequence can be complementary to a first target RNA region and thesecond sequence can be complementary to a second target RNA region. Thefirst and second sequences of the iRNA agent can be on different RNAstrands, and the mismatch between the first and second sequences can beless than 50%, 40%, 30%, 20%, 10%, 5%, or 1%. The first and secondsequences of the iRNA agent are on the same RNA strand, and in a relatedembodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNAagent can be in bimolecular form. The first and second sequences of theiRNA agent can be fully complementary to each other.

The first target RNA region can be encoded by a first gene and thesecond target RNA region can encoded by a second gene, or the first andsecond target RNA regions can be different regions of an RNA from asingle gene. The first and second sequences can differ by at least 1nucleotide.

The first and second target RNA regions can be on transcripts encoded byfirst and second sequence variants, e.g., first and second alleles, of agene. The sequence variants can be mutations, or polymorphisms, forexample. The first target RNA region can include a nucleotidesubstitution, insertion, or deletion relative to the second target RNAregion, or the second target RNA region can a mutant or variant of thefirst target region.

The first and second target RNA regions can comprise viral or human RNAregions. The first and second target RNA regions can also be on varianttranscripts of an oncogene or include different mutations of a tumorsuppressor gene transcript. In addition, the first and second target RNAregions can correspond to hot-spots for genetic variation.

The compositions of the invention can include mixtures of iRNA agentmolecules. For example, one iRNA agent can contain a first sequence anda second sequence sufficiently complementary to each other to hybridize,and in addition the first sequence is complementary to a first targetRNA region and the second sequence is complementary to a second targetRNA region. The mixture can also include at least one additional iRNAagent variety that includes a third sequence and a fourth sequencesufficiently complementary to each other to hybridize, and where thethird sequence is complementary to a third target RNA region and thefourth sequence is complementary to a fourth target RNA region. Inaddition, the first or second sequence can be sufficiently complementaryto the third or fourth sequence to be capable of hybridizing to eachother. The first and second sequences can be on the same or differentRNA strands, and the third and fourth sequences can be on the same ordifferent RNA strands.

The target RNA regions can be variant sequences of a viral or human RNA,and in certain embodiments, at least two of the target RNA regions canbe on variant transcripts of an oncogene or tumor suppressor gene. Thetarget RNA regions can correspond to genetic hot-spots.

Methods of making an iRNA agent composition can include obtaining orproviding information about a region of an RNA of a target gene (e.g., aviral or human gene, or an oncogene or tumor suppressor, e.g., p53),where the region has high variability or mutational frequency (e.g., inhumans). In addition, information about a plurality of RNA targetswithin the region can be obtained or provided, where each RNA targetcorresponds to a different variant or mutant of the gene (e.g., a regionincluding the codon encoding p53 248Q and/or p53 249S). The iRNA agentcan be constructed such that a first sequence is complementary to afirst of the plurality of variant RNA targets (e.g., encoding 249Q) anda second sequence is complementary to a second of the plurality ofvariant RNA targets (e.g., encoding 249S), and the first and secondsequences can be sufficiently complementary to hybridize.

Sequence analysis, e.g., to identify common mutants in the target gene,can be used to identify a region of the target gene that has highvariability or mutational frequency. A region of the target gene havinghigh variability or mutational frequency can be identified by obtainingor providing genotype information about the target gene from apopulation.

Expression of a target gene can be modulated, e.g., downregulated orsilenced, by providing an iRNA agent that has a first sequence and asecond sequence sufficiently complementary to each other to hybridize.In addition, the first sequence can be complementary to a first targetRNA region and the second sequence can be complementary to a secondtarget RNA region.

An iRNA agent can include a first sequence complementary to a firstvariant RNA target region and a second sequence complementary to asecond variant RNA target region. The first and second variant RNAtarget regions can correspond to first and second variants or mutants ofa target gene, e.g., viral gene, tumor suppressor or oncogene. The firstand second variant target RNA regions can include allelic variants,mutations (e.g., point mutations), or polymorphisms of the target gene.The first and second variant RNA target regions can correspond togenetic hot-spots.

A plurality of iRNA agents (e.g., a panel or bank) can be provided.

OTHER EMBODIMENTS

In yet another embodiment, iRNAs agents are produced in a cell in vivo,e.g., from exogenous DNA templates that are delivered into the cell. Forexample, the DNA templates can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of a iRNA agent and one that produces atranscript that includes the bottom strand of a iRNA agent. When thetemplates are transcribed, the iRNA agent is produced, and processedinto siRNA agent fragments that mediate gene silencing.

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end.

Antagomirs may be used to efficiently silence endogenous miRNAs therebypreventing miRNA-induced gene silencing. An example ofantagomir-mediated miRNA silencing is the silencing of miR-122,described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which isexpressly incorporated by reference herein, in its entirety.

Decoy Oligonucleotides

Because transcription factors can recognize their relatively shortbinding sequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides may be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety.

Antisense Oligonucleotides

Antisense oligonucleotides are single strands of DNA or RNA that are atleast partially complementary to a chosen sequence. In the case ofantisense RNA, they prevent translation of complementary RNA strands bybinding to it. Antisense DNA can also be used to target a specific,complementary (coding or non-coding) RNA. If binding takes place, theDNA/RNA hybrid can be degraded by the enzyme RNase H. Examples of theutilization of antisense oligonucleotides may be found in Dias et al.,Mol. Cancer Ther., 2002, 1: 347-355, which is expressly incorporated byreference herein, in its entirety.

Aptamers

Aptamers are nucleic acid molecules that bind a specific target moleculeor molecules. Aptamers may be RNA or DNA based, and may include ariboswitch. A riboswitch is a part of an mRNA molecule that can directlybind a small target molecule, and whose binding of the target affectsthe gene's activity. Thus, an mRNA that contains a riboswitch isdirectly involved in regulating its own activity, depending on thepresence or absence of its target molecule.

Physiological Effects

The iRNA agents described herein can be designed such that determiningtherapeutic toxicity is made easier by the complementarity of the iRNAagent with both a human and a non-human animal sequence. By thesemethods, an iRNA agent can consist of a sequence that is fullycomplementary to a nucleic acid sequence from a human and a nucleic acidsequence from at least one non-human animal, e.g., a non-human mammal,such as a rodent, ruminant or primate. For example, the non-human mammalcan be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus,Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence ofthe iRNA agent could be complementary to sequences within homologousgenes, e.g., oncogenes or tumor suppressor genes, of the non-humanmammal and the human. By determining the toxicity of the iRNA agent inthe non-human mammal, one can extrapolate the toxicity of the iRNA agentin a human. For a more strenuous toxicity test, the iRNA agent can becomplementary to a human and more than one, e.g., two or three or more,non-human animals.

The methods described herein can be used to correlate any physiologicaleffect of an iRNA agent on a human, e.g., any unwanted effect, such as atoxic effect, or any positive, or desired effect.

Increasing Cellular Uptake of dsiRNAs

A method of the invention that includes administering an iRNA agent anda drug that affects the uptake of the iRNA agent into the cell. The drugcan be administered before, after, or at the same time that the iRNAagent is administered. The drug can be covalently linked to the iRNAagent. The drug can be, for example, a lipopolysaccharid, an activatorof p38 MAP kinase, or an activator of NF-κB. The drug can have atransient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, forexample, by disrupting the cell's cytoskeleton, e.g., by disrupting thecell's microtubules, microfilaments, and/or intermediate filaments. Thedrug can be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

The drug can also increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary drug's thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a secondagent. For example, an iRNA agent used to treat a particular disordercan be coupled to a second therapeutic agent, e.g., an agent other thanthe iRNA agent. The second therapeutic agent can be one which isdirected to the treatment of the same disorder. For example, in the caseof an iRNA used to treat a disorder characterized by unwanted cellproliferation, e.g., cancer, the iRNA agent can be coupled to a secondagent which has an anti-cancer effect. For example, it can be coupled toan agent which stimulates the immune system, e.g., a CpG motif, or moregenerally an agent that activates a toll-like receptor and/or increasesthe production of gamma interferon.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods.Exemplary methods include: organic synthesis and RNA cleavage, e.g., invitro cleavage.

Organic Synthesis

An iRNA can be made by separately synthesizing each respective strand ofa double-stranded RNA molecule. The component strands can then beannealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB(Uppsala Sweden), can be used to produce a large amount of a particularRNA strand for a given iRNA. The OligoPilotII reactor can efficientlycouple a nucleotide using only a 1.5 molar excess of a phosphoramiditenucleotide. To make an RNA strand, ribonucleotides amidites are used.Standard cycles of monomer addition can be used to synthesize the 21 to23 nucleotide strand for the iRNA. Typically, the two complementarystrands are produced separately and then annealed, e.g., after releasefrom the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. Thecomplementary of the species to a particular target gene can beprecisely specified. For example, the species may be complementary to aregion that includes a polymorphism, e.g., a single nucleotidepolymorphism. Further the location of the polymorphism can be preciselydefined. In some embodiments, the polymorphism is located in an internalregion, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of thetermini.

dsiRNA Cleavage

iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can bemediated in vitro or in vivo. For example, to produce iRNAs by cleavagein vitro, the following method can be used:

In vitro transcription. dsiRNA is produced by transcribing a nucleicacid (DNA) segment in both directions. For example, the HiScribe™ RNAitranscription kit (New England Biolabs) provides a vector and a methodfor producing a dsiRNA for a nucleic acid segment that is cloned intothe vector at a position flanked on either side by a T7 promoter.Separate templates are generated for T7 transcription of the twocomplementary strands for the dsiRNA. The templates are transcribed invitro by addition of T7 RNA polymerase and dsiRNA is produced. Similarmethods using PCR and/or other RNA polymerases (e.g., T3 or SP6polymerase) can also be used. In one embodiment, RNA generated by thismethod is carefully purified to remove endotoxins that may contaminatepreparations of the recombinant enzymes.

In vitro cleavage. dsiRNA is cleaved in vitro into iRNAs, for example,using a Dicer or comparable RNAse III-based activity. For example, thedsiRNA can be incubated in an in vitro extract from Drosophila or usingpurified components, e.g., a purified RNAse or RISC complex (RNA-inducedsilencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9. and Hammond Science 2001 Aug. 10; 293 (5532):1146-50.

dsiRNA cleavage generally produces a plurality of iRNA species, eachbeing a particular 21 to 23 nt fragment of a source dsiRNA molecule. Forexample, iRNAs that include sequences complementary to overlappingregions and adjacent regions of a source dsiRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Formulation

The iRNA agents described herein can be formulated for administration toa subject.

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It may be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration (see, below).

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA that can mediated RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA). Forexample, a iRNA composition for the treatment of a viral disease, e.g.,HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, a iRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

Exemplary formulations are discussed below:

Liposomes

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It may be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA s agents, and such practice is within the invention. AniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a siRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, or precursor thereof) preparation can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a iRNA can be prepared by a variety of methods.

In one example, the lipid component of a liposome is dissolved in adetergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNApreparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA andcondense around the iRNA to form a liposome. After condensation, thedetergent is removed, e.g., by dialysis, to yield a liposomalpreparation of iRNA.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Further description of methods for producing stable polynucleotidedelivery vehicles, which incorporate a polynucleotide/cationic lipidcomplex as structural components of the delivery vehicle, are describedin, e.g., WO 96/37194. Liposome formation can also include one or moreaspects of exemplary methods described in Felgner, P. L. et al., Proc.Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S.Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson,et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl.Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; andFukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques forpreparing lipid aggregates of appropriate size for use as deliveryvehicles include sonication and freeze-thaw plus extrusion (see, e.g.,Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidizationcan be used when consistently small (50 to 200 nm) and relativelyuniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta775:169, 1984). These methods are readily adapted to packaging iRNApreparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged, entrap nucleicacid molecules rather than complex with them. Since both the nucleicacid molecules and the lipid are similarly charged, repulsion ratherthan complex formation occurs. Nevertheless, some nucleic acid moleculesare entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA (see, e.g., Felgner,P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S.Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA, into the skin. In some implementations,liposomes are used for delivering iRNA to epidermal cells and also toenhance the penetration of iRNA into dermal tissues, e.g., into skin.For example, the liposomes can be applied topically. Topical delivery ofdrugs formulated as liposomes to the skin has been documented (see,e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino,R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T.et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176,1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527,1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNA can bedelivered, for example, subcutaneously by infection in order to deliveriRNA to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Surfactants

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It may be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes (see above). iRNA (or aprecursor, e.g., a larger dsiRNA which can be processed into a iRNA, ora DNA which encodes a iRNA or precursor) compositions can include asurfactant. In one embodiment, the iRNA is formulated as an emulsionthat includes a surfactant. The most common way of classifying andranking the properties of the many different types of surfactants, bothnatural and synthetic, is by the use of the hydrophile/lipophile balance(HLB). The nature of the hydrophilic group provides the most usefulmeans for categorizing the different surfactants used in formulations(Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., NewYork, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical products and are usable over a wide range of pH values.In general their HLB values range from 2 to about 18 depending on theirstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters.Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and Other Membranous Formulations

For ease of exposition the micelles and other formulations, compositionsand methods in this section are discussed largely with regard tounmodified iRNA agents. It may be understood, however, that thesemicelles and other formulations, compositions and methods can bepracticed with other iRNA agents, e.g., modified iRNA agents, and suchpractice is within the invention. The iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof)) composition can be provided as amicellar formulation. “Micelles” are defined herein as a particular typeof molecular assembly in which amphipathic molecules are arranged in aspherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the iRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe iRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the iRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

Particles

For ease of exposition the particles, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It may be understood, however, that these particles,formulations, compositions and methods can be practiced with other iRNAagents, e.g., modified iRNA agents, and such practice is within theinvention. In another embodiment, an iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNAagent which can be processed into a siRNA agent, or a DNA which encodesan iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof) preparations may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques. See below for further description.

Sustained-Release Formulations. An iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNAagent which can be processed into a siRNA agent, or a DNA which encodesan iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof) described herein can be formulated for controlled,e.g., slow release. Controlled release can be achieved by disposing theiRNA within a structure or substance which impedes its release. E.g.,iRNA can be disposed within a porous matrix or in an erodable matrix,either of which allow release of the iRNA over a period of time.

Polymeric particles, e.g., polymeric in microparticles can be used as asustained-release reservoir of iRNA that is taken up by cells onlyreleased from the microparticle through biodegradation. The polymericparticles in this embodiment should therefore be large enough topreclude phagocytosis (e.g., larger than 10 μm or larger than 20 μm).Such particles can be produced by the same methods to make smallerparticles, but with less vigorous mixing of the first and secondemulsions. That is to say, a lower homogenization speed, vortex mixingspeed, or sonication setting can be used to obtain particles having adiameter around 100 μm rather than 10 μm. The time of mixing also can bealtered.

Larger microparticles can be formulated as a suspension, a powder, or animplantable solid, to be delivered by intramuscular, subcutaneous,intradermal, intravenous, or intraperitoneal injection; via inhalation(intranasal or intrapulmonary); orally; or by implantation. Theseparticles are useful for delivery of any iRNA when slow release over arelatively long term is desired. The rate of degradation, andconsequently of release, varies with the polymeric formulation.

Microparticles may include pores, voids, hollows, defects or otherinterstitial spaces that allow the fluid suspension medium to freelypermeate or perfuse the particulate boundary. For example, theperforated microstructures can be used to form hollow, porous spraydried microspheres.

Polymeric particles containing iRNA (e.g., a siRNA) can be made using adouble emulsion technique, for instance. First, the polymer is dissolvedin an organic solvent. A polymer may be polylactic-co-glycolic acid(PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or75:25. Next, a sample of nucleic acid suspended in aqueous solution isadded to the polymer solution and the two solutions are mixed to form afirst emulsion. The solutions can be mixed by vortexing or shaking, andin the mixture can be sonicated. Any method by which the nucleic acidreceives the least amount of damage in the form of nicking, shearing, ordegradation, while still allowing the formation of an appropriateemulsion is possible. For example, acceptable results can be obtainedwith a Vibra-cell model VC-250 sonicator with a ⅛″ microtip probe, atsetting #3.

Spray Drying

An iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent,(e.g., a precursor, e.g., a larger iRNA agent which can be processedinto a siRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, or precursor thereof)) canbe prepared by spray drying. Spray dried iRNA can be administered to asubject or be subjected to further formulation. A pharmaceuticalcomposition of iRNA can be prepared by spray drying a homogeneousaqueous mixture that includes a iRNA under conditions sufficient toprovide a dispersible powdered composition, e.g., a pharmaceuticalcomposition. The material for spray drying can also include one or moreof: a pharmaceutically acceptable excipient, or adispersibility-enhancing amount of a physiologically acceptable,water-soluble protein. The spray-dried product can be a dispersiblepowder that includes the iRNA.

Spray drying is a process that converts a liquid or slurry material to adried particulate form. Spray drying can be used to provide powderedmaterial for various administrative routes including inhalation. See,for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols:Physical and Biological Basis for Therapy, A. J. Hickey, ed. MarcelDekkar, New York, 1996.

Spray drying can include atomizing a solution, emulsion, or suspensionto form a fine mist of droplets and drying the droplets. The mist can beprojected into a drying chamber (e.g., a vessel, tank, tubing, or coil)where it contacts a drying gas. The mist can include solid or liquidpore forming agents. The solvent and pore forming agents evaporate fromthe droplets into the drying gas to solidify the droplets,simultaneously forming pores throughout the solid. The solid (typicallyin a powder, particulate form) then is separated from the drying gas andcollected.

Spray drying includes bringing together a highly dispersed liquid, and asufficient volume of air (e.g., hot air) to produce evaporation anddrying of the liquid droplets. The preparation to be spray dried can beany solution, course suspension, slurry, colloidal dispersion, or pastethat may be atomized using the selected spray drying apparatus.Typically, the feed is sprayed into a current of warm filtered air thatevaporates the solvent and conveys the dried product to a collector. Thespent air is then exhausted with the solvent. Several different types ofapparatus may be used to provide the desired product. For example,commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. caneffectively produce particles of desired size.

Spray-dried powdered particles can be approximately spherical in shape,nearly uniform in size and frequently hollow. There may be some degreeof irregularity in shape depending upon the incorporated medicament andthe spray drying conditions. In many instances the dispersion stabilityof spray-dried microspheres appears to be more effective if an inflatingagent (or blowing agent) is used in their production. Certainembodiments may comprise an emulsion with an inflating agent as thedisperse or continuous phase (the other phase being aqueous in nature).An inflating agentmay be dispersed with a surfactant solution, using,for instance, a commercially available microfluidizer at a pressure ofabout 5000 to 15,000 psi. This process forms an emulsion, which may bestabilized by an incorporated surfactant, typically comprising submicrondroplets of water immiscible blowing agent dispersed in an aqueouscontinuous phase. The formation of such dispersions using this and othertechniques are common and well known to those in the art. The blowingagent may be a fluorinated compound (e.g., perfluorohexane,perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) whichvaporizes during the spray-drying process, leaving behind generallyhollow, porous aerodynamically light microspheres. As will be discussedin more detail below, other suitable blowing agents include chloroform,freons, and hydrocarbons. Nitrogen gas and carbon dioxide are alsocontemplated as a suitable blowing agent.

Although the perforated microstructures may be formed using a blowingagent as described above, it will be appreciated that, in someinstances, no blowing agent is required and an aqueous dispersion of themedicament and surfactant(s) are spray dried directly. In such cases,the formulation may be amenable to process conditions (e.g., elevatedtemperatures) that generally lead to the formation of hollow, relativelyporous microparticles. Moreover, the medicament may possess specialphysicochemical properties (e.g., high crystallinity, elevated meltingtemperature, surface activity, etc.) that make it particularly suitablefor use in such techniques.

The perforated microstructures may optionally be associated with, orcomprise, one or more surfactants. Moreover, miscible surfactants mayoptionally be combined with the suspension medium liquid phase. It willbe appreciated by those skilled in the art that the use of surfactantsmay further increase dispersion stability, simplify formulationprocedures or increase bioavailability upon administration. Of coursecombinations of surfactants, including the use of one or more in theliquid phase and one or more associated with the perforatedmicrostructures are contemplated as being within the scope of theinvention. By “associated with or comprise” it is meant that thestructural matrix or perforated microstructure may incorporate, adsorb,absorb, be coated with or be formed by the surfactant.

Surfactants suitable for use include any compound or composition thataids in the formation and maintenance of the stabilized respiratorydispersions by forming a layer at the interface between the structuralmatrix and the suspension medium. The surfactant may comprise a singlecompound or any combination of compounds, such as in the case ofco-surfactants. Particularly certain surfactants are substantiallyinsoluble in the propellant, nonfluorinated, and selected from the groupconsisting of saturated and unsaturated lipids, nonionic detergents,nonionic block copolymers, ionic surfactants, and combinations of suchagents. It may be emphasized that, in addition to the aforementionedsurfactants, suitable (i.e., biocompatible) fluorinated surfactants arecompatible with the teachings herein and may be used to provide thedesired stabilized preparations.

Lipids, including phospholipids, from both natural and synthetic sourcesmay be used in varying concentrations to form a structural matrix.Generally, compatible lipids comprise those that have a gel to liquidcrystal phase transition greater than about 40° C. In certainembodiments, the incorporated lipids are relatively long chain (i.e.,C₆-C₂₂) saturated lipids and may comprise phospholipids. Exemplaryphospholipids useful in the disclosed stabilized preparations compriseegg phosphatidylcholine, dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline,disteroylphosphatidylcholine, short-chain phosphatidylcholines,phosphatidylethanolamine, dioleylphosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol,glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid,cardiolipin; lipids bearing polymer chains such as, polyethylene glycol,chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearingsulfonated mono-, di-, and polysaccharides; fatty acids such as palmiticacid, stearic acid, and oleic acid; cholesterol, cholesterol esters, andcholesterol hemisuccinate. Due to their excellent biocompatibilitycharacteristics, phospholipids and combinations of phospholipids andpoloxamers are particularly suitable for use in the stabilizeddispersions disclosed herein.

Compatible nonionic detergents comprise: sorbitan esters includingsorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitanmonooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitanmonolaurate, and polyoxyethylene (20) sorbitan monooleate, oleylpolyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, laurylpolyoxyethylene (4) ether, glycerol esters, and sucrose esters. Othersuitable nonionic detergents can be easily identified using McCutcheon'sEmulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Certainblock copolymers include diblock and triblock copolymers ofpolyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic®F68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionicsurfactants such as sodium sulfosuccinate, and fatty acid soaps may alsobe utilized. In certain embodiments, the microstructures may compriseoleic acid or its alkali salt.

In addition to the aforementioned surfactants, cationic surfactants orlipids may be used, especially in the case of delivery of an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,e.g., a larger iRNA agent which can be processed into a siRNA agent, ora DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent,or siRNA agent, or precursor thereof). Examples of suitable cationiclipids include: DOTMA,N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP,1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB,1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationicamino acids such as polylysine, and polyarginine are also contemplated.

For the spraying process, such spraying methods as rotary atomization,pressure atomization and two-fluid atomization can be used. Examples ofthe devices used in these processes include “Parubisu [phoneticrendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”,manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray DrierL-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,”manufactured by Okawara Kakoki Co., can be used for the method ofspraying using rotary-disk atomizer.

While no particular restrictions are placed on the gas used to dry thesprayed material, it is recommended to use air, nitrogen gas or an inertgas. The temperature of the inlet of the gas used to dry the sprayedmaterials such that it does not cause heat deactivation of the sprayedmaterial. The range of temperatures may vary between about 50° C. toabout 200° C., for example, between about 50° C. and 100° C. Thetemperature of the outlet gas used to dry the sprayed material, may varybetween about 0° C. and about 150° C., for example, between 0° C. and90° C., and for example between 0° C. and 60° C.

The spray drying is done under conditions that result in substantiallyamorphous powder of homogeneous constitution having a particle size thatis respirable, a low moisture content and flow characteristics thatallow for ready aerosolization. In some cases, the particle size of theresulting powder is such that more than about 98% of the mass is inparticles having a diameter of about 10 μm or less with about 90% of themass being in particles having a diameter less than 5 μm. Alternatively,about 95% of the mass will have particles with a diameter of less than10 μm with about 80% of the mass of the particles having a diameter ofless than 5 μm.

The dispersible pharmaceutical-based dry powders that include the iRNApreparation may optionally be combined with pharmaceutical carriers orexcipients which are suitable for respiratory and pulmonaryadministration. Such carriers may serve simply as bulking agents when itis desired to reduce the iRNA concentration in the powder which is beingdelivered to a patient, but may also serve to enhance the stability ofthe iRNA compositions and to improve the dispersibility of the powderwithin a powder dispersion device in order to provide more efficient andreproducible delivery of the iRNA and to improve handlingcharacteristics of the iRNA such as flowability and consistency tofacilitate manufacturing and powder filling.

Such carrier materials may be combined with the drug prior to spraydrying, i.e., by adding the carrier material to the purified bulksolution. In that way, the carrier particles will be formedsimultaneously with the drug particles to produce a homogeneous powder.Alternatively, the carriers may be separately prepared in a dry powderform and combined with the dry powder drug by blending. The powdercarriers will usually be crystalline (to avoid water absorption), butmight in some cases be amorphous or mixtures of crystalline andamorphous. The size of the carrier particles may be selected to improvethe flowability of the drug powder, typically being in the range from 25μm to 100 μm. A carrier material may be crystalline lactose having asize in the above-stated range.

Powders prepared by any of the above methods will be collected from thespray dryer in a conventional manner for subsequent use. For use aspharmaceuticals and other purposes, it will frequently be desirable todisrupt any agglomerates which may have formed by screening or otherconventional techniques. For pharmaceutical uses, the dry powderformulations will usually be measured into a single dose, and the singledose sealed into a package. Such packages are particularly useful fordispersion in dry powder inhalers, as described in detail below.Alternatively, the powders may be packaged in multiple-dose containers.

Methods for spray drying hydrophobic and other drugs and components aredescribed in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602;and 4,486,435. Bloch and Speison (1983) Pharm. Acta Helv 58:14-22teaches spray drying of hydrochlorothiazide and chlorthalidone(lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) inazeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A numberof Japanese Patent application Abstracts relate to spray drying ofhydrophilic-hydrophobic product combinations, including JP 806766; JP7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP4036233. Other foreign patent publications relevant to spray dryinghydrophilic-hydrophobic product combinations include FR 2594693; DE2209477; and WO 88/07870.

Lyophilization

An iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent,(e.g., a precursor, e.g., a larger iRNA agent which can be processedinto a siRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, or precursor thereof)preparation can be made by lyophilization. Lyophilization is afreeze-drying process in which water is sublimed from the compositionafter it is frozen. The particular advantage associated with thelyophilization process is that biologicals and pharmaceuticals that arerelatively unstable in an aqueous solution can be dried without elevatedtemperatures (thereby eliminating the adverse thermal effects), and thenstored in a dry state where there are few stability problems. Withrespect to the instant invention such techniques are particularlycompatible with the incorporation of nucleic acids in perforatedmicrostructures without compromising physiological activity. Methods forproviding lyophilized particulates are known to those of skill in theart and it would clearly not require undue experimentation to providedispersion compatible microstructures in accordance with the teachingsherein. Accordingly, to the extent that lyophilization processes may beused to provide microstructures having the desired porosity and size,they are conformance with the teachings herein and are expresslycontemplated as being within the scope of the instant invention.

Genes

In one aspect, the invention features, a method of treating a subject atrisk for or afflicted with a disease that may benefit from theadministration of the iRNA agent of the invention. The method comprisesadministering the iRNA agent of the invention to a subject in needthereof, thereby treating the subject. The iRNA agent that isadministered will depend on the disease being treated.

In certain embodiments, the iRNA agent silences a growth factor orgrowth factor receptor gene, a kinase, e.g., a protein tyrosine, serineor threonine kinase gene, an adaptor protein gene, a gene encoding a Gprotein superfamily molecule, or a gene encoding a transcription factor.

In some embodiments the iRNA agent silences the PDGF beta gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted PDGF beta expression, e.g., testicular andlung cancers.

In some embodiments the iRNA agent silences the Erb-B gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted Erb-B expression, e.g., breast cancer.

In some embodiments the iRNA agent silences the Src gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted Src expression, e.g., colon cancers.

In some embodiments the iRNA agent silences the CRK gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted CRK expression, e.g., colon and lung cancers.

In some embodiments the iRNA agent silences the GRB2 gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted GRB2 expression, e.g., squamous cellcarcinoma.

In another embodiment the iRNA agent silences the RAS gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted RAS expression, e.g., pancreatic, colon andlung cancers, and chronic leukemia.

In another embodiment the iRNA agent silences the MEKK gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted MEKK expression, e.g., squamous cellcarcinoma, melanoma or leukemia.

In another embodiment the iRNA agent silences the JNK gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted JNK expression, e.g., pancreatic or breastcancers.

In some embodiments the iRNA agent silences the RAF gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted RAF expression, e.g., lung cancer or leukemia.

In some embodiments the iRNA agent silences the Erk1/2 gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted Erk1/2 expression, e.g., lung cancer.

In another embodiment the iRNA agent silences the PCNA(p21) gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted PCNA expression, e.g., lung cancer.

In some embodiments the iRNA agent silences the MYB gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted MYB expression, e.g., colon cancer or chronicmyelogenous leukemia.

In some embodiments the iRNA agent silences the c-MYC gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma orneuroblastoma.

In another embodiment the iRNA agent silences the JUN gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted JUN expression, e.g., ovarian, prostate orbreast cancers.

In another embodiment the iRNA agent silences the FOS gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted FOS expression, e.g., skin or prostatecancers.

In some embodiments the iRNA agent silences the BCL-2 gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted BCL-2 expression, e.g., lung or prostatecancers or Non-Hodgkin lymphoma.

In some embodiments the iRNA agent silences the Cyclin D gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted Cyclin D expression, e.g., esophageal andcolon cancers.

In some embodiments the iRNA agent silences the VEGF gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted VEGF expression, e.g., esophageal and coloncancers.

In some embodiments the iRNA agent silences the EGFR gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted EGFR expression, e.g., breast cancer.

In another embodiment the iRNA agent silences the Cyclin A gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Cyclin A expression, e.g., lung and cervicalcancers.

In another embodiment the iRNA agent silences the Cyclin E gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Cyclin E expression, e.g., lung and breastcancers.

In another embodiment the iRNA agent silences the WNT-1 gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.

In another embodiment the iRNA agent silences the beta-catenin gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted beta-catenin expression, e.g., adenocarcinomaor hepatocellular carcinoma.

In another embodiment the iRNA agent silences the c-MET gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted c-MET expression, e.g., hepatocellularcarcinoma.

In another embodiment the iRNA agent silences the PKC gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted PKC expression, e.g., breast cancer.

In some embodiments the iRNA agent silences the NFKB gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted NFKB expression, e.g., breast cancer.

In some embodiments the iRNA agent silences the STAT3 gene, and thus canbe used to treat a subject having or at risk for a disordercharacterized by unwanted STAT3 expression, e.g., prostate cancer.

In another embodiment the iRNA agent silences the survivin gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted survivin expression, e.g., cervical orpancreatic cancers.

In another embodiment the iRNA agent silences the Her2/Neu gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Her2/Neu expression, e.g., breast cancer.

In another embodiment the iRNA agent silences the topoisomerase I gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted topoisomerase I expression, e.g., ovarian andcolon cancers.

In some embodiments the iRNA agent silences the topoisomerase II alphagene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted topoisomerase II expression, e.g.,breast and colon cancers.

In some embodiments the iRNA agent silences mutations in the p73 gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted p73 expression, e.g., colorectaladenocarcinoma.

In some embodiments the iRNA agent silences mutations in the p21(WAF1/CIP1) gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted p21 (WAF1/CIP1)expression, e.g., liver cancer.

In some embodiments the iRNA agent silences mutations in the p27 (KIP1)gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted p27 (KIP1) expression, e.g., livercancer.

In some embodiments the iRNA agent silences mutations in the PPM1D gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted PPM1D expression, e.g., breast cancer.

In some embodiments the iRNA agent silences mutations in the RAS gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted RAS expression, e.g., breast cancer.

In another embodiment the iRNA agent silences mutations in the caveolinI gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted caveolin I expression, e.g.,esophageal squamous cell carcinoma.

In another embodiment the iRNA agent silences mutations in the MIB Igene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted MIB I expression, e.g., male breastcarcinoma (MBC).

In another embodiment the iRNA agent silences mutations in the MTAIgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted MTAI expression, e.g., ovariancarcinoma.

In another embodiment the iRNA agent silences mutations in the M68 gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted M68 expression, e.g., human adenocarcinomas ofthe esophagus, stomach, colon, and rectum.

In certain embodiments the iRNA agent silences mutations in tumorsuppressor genes, and thus can be used as a method to promote apoptoticactivity in combination with chemotherapeutics.

In some embodiments the iRNA agent silences mutations in the p53 tumorsuppressor gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted p53 expression, e.g., gallbladder, pancreatic and lung cancers.

In some embodiments the iRNA agent silences mutations in the p53 familymember DN-p63, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted DN-p63 expression, e.g.,squamous cell carcinoma

In some embodiments the iRNA agent silences mutations in the pRb tumorsuppressor gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted pRb expression, e.g., oralsquamous cell carcinoma

In some embodiments the iRNA agent silences mutations in the APC1 tumorsuppressor gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted APC1 expression, e.g.,colon cancer.

In some embodiments the iRNA agent silences mutations in the BRCA1 tumorsuppressor gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted BRCA1 expression, e.g.,breast cancer.

In some embodiments the iRNA agent silences mutations in the PTEN tumorsuppressor gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted PTEN expression, e.g.,hamartomas, gliomas, and prostate and endometrial cancers.

In some embodiments the iRNA agent silences MLL fusion genes, e.g.,MLL-AF9, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted MLL fusion gene expression, e.g.,acute leukemias.

In another embodiment the iRNA agent silences the BCR/ABL fusion gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted BCR/ABL fusion gene expression, e.g., acuteand chronic leukemias.

In another embodiment the iRNA agent silences the TEL/AML1 fusion gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted TEL/AML1 fusion gene expression, e.g.,childhood acute leukemia.

In another embodiment the iRNA agent silences the EWS/FLI1 fusion gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted EWS/FLI1 fusion gene expression, e.g., EwingSarcoma.

In another embodiment the iRNA agent silences the TLS/FUS1 fusion gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted TLS/FUS1 fusion gene expression, e.g., Myxoidliposarcoma.

In another embodiment the iRNA agent silences the PAX3/FKHR fusion gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoidliposarcoma.

In another embodiment the iRNA agent silences the AML1/ETO fusion gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted AML1/ETO fusion gene expression, e.g., acuteleukemia.

Diseases

Angiogenesis

In another aspect, the invention features, a method of treating asubject, e.g., a human, at risk for or afflicted with a disease ordisorder that may benefit by angiogenesis inhibition, e.g., cancer. Themethod comprises administering the iRNA agent of the invention to asubject in need thereof, thereby treating the subject. The iRNA agentthat is administered will depend on the type of angiogenesis-relatedgene being treated.

In some embodiments the iRNA agent silences the alpha v-integrin gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted alpha V integrin, e.g., brain tumors or tumorsof epithelial origin.

In some embodiments the iRNA agent silences the Flt-1 receptor gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Flt-1 receptors, eg. cancer and rheumatoidarthritis.

In some embodiments the iRNA agent silences the tubulin gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted tubulin, eg. cancer and retinalneovascularization.

In some embodiments the iRNA agent silences the tubulin gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted tubulin, eg. cancer and retinalneovascularization.

Viral Diseases

In yet another aspect, the invention features a method of treating asubject infected with a virus or at risk for or afflicted with adisorder or disease associated with a viral infection. The methodcomprises administering an iRNA agent of the invention to a subject inneed thereof, thereby treating the subject. The iRNA agent that isadministered will depend on the type of viral disease being treated. Insome embodiments, the nucleic acid may target a viral gene. In otherembodiments, the nucleic acid may target a host gene.

Thus, the invention provides for a method of treating patients infectedby the Human Papilloma Virus (HPV) or at risk for or afflicted with adisorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% ofcervical carcinomas and thus an antiviral therapy is an attractivemethod to treat these cancers and other symptoms of viral infection. Insome embodiments, the expression of a HPV gene is reduced. In anotherembodiment, the HPV gene is one of the group of E2, E6, or E7. In someembodiments the expression of a human gene that is required for HPVreplication is reduced.

The invention also includes a method of treating patients infected bythe Human Immunodeficiency Virus (HIV) or at risk for or afflicted witha disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome(AIDS). In some embodiments, the expression of a HIV gene is reduced. Inanother embodiment, the HIV gene is CCR5, Gag, or Rev. In someembodiments the expression of a human gene that is required for HIVreplication is reduced. In another embodiment, the gene is CD4 orTsg101.

The invention also includes a method for treating patients infected bythe Hepatitis B Virus (HBV) or at risk for or afflicted with a disordermediated by HBV, e.g., cirrhosis and heptocellular carcinoma. In someembodiments, the expression of a HBV gene is reduced. In anotherembodiment, the targeted HBV gene encodes one of the group of the tailregion of the HBV core protein, the pre-cregious (pre-c) region, or thecregious (c) region. In another embodiment, a targeted HBV-RNA sequenceis comprised of the poly(A) tail. In certain embodiment the expressionof a human gene that is required for HBV replication is reduced.

The invention also provides for a method of treating patients infectedby the Hepatitis A Virus (HAV), or at risk for or afflicted with adisorder mediated by HAV. In some embodiments the expression of a humangene that is required for HAV replication is reduced.

The present invention provides for a method of treating patientsinfected by the Hepatitis C Virus (HCV), or at risk for or afflictedwith a disorder mediated by HCV, e.g., cirrhosis. In some embodiments,the expression of a HCV gene is reduced. In another embodiment theexpression of a human gene that is required for HCV replication isreduced.

The present invention also provides for a method of treating patientsinfected by the any of the group of Hepatitis Viral strains comprisinghepatitis D, E, F, G, or H, or patients at risk for or afflicted with adisorder mediated by any of these strains of hepatitis. In someembodiments, the expression of a Hepatitis, D, E, F, G, or H gene isreduced. In another embodiment the expression of a human gene that isrequired for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected bythe Respiratory Syncytial Virus (RSV) or at risk for or afflicted with adisorder mediated by RSV, e.g, lower respiratory tract infection ininfants and childhood asthma, pneumonia and other complications, e.g.,in the elderly. In some embodiments, the expression of a RSV gene isreduced. In another embodiment, the targeted HBV gene encodes one of thegroup of genes N, L, or P. In some embodiments the expression of a humangene that is required for RSV replication is reduced.

Methods of the invention provide for treating patients infected by theHerpes Simplex Virus (HSV) or at risk for or afflicted with a disordermediated by HSV, e.g, genital herpes and cold sores as well aslife-threatening or sight-impairing disease mainly in immunocompromisedpatients. In some embodiments, the expression of a HSV gene is reduced.In another embodiment, the targeted HSV gene encodes DNA polymerase orthe helicase-primase. In some embodiments the expression of a human genethat is required for HSV replication is reduced.

The invention also provides a method for treating patients infected bythe herpes Cytomegalovirus (CMV) or at risk for or afflicted with adisorder mediated by CMV, e.g., congenital virus infections andmorbidity in immunocompromised patients. In some embodiments, theexpression of a CMV gene is reduced. In some embodiments the expressionof a human gene that is required for CMV replication is reduced.

Methods of the invention also provide for a method of treating patientsinfected by the herpes Epstein Barr Virus (EBV) or at risk for orafflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma,non-Hodgkin lymphoma, and Hodgkin disease. In some embodiments, theexpression of a EBV gene is reduced. In some embodiments the expressionof a human gene that is required for EBV replication is reduced.

Methods of the invention also provide for treating patients infected byKaposi's Sarcoma-associated Herpes Virus (KSHV), also called humanherpesvirus 8, or patients at risk for or afflicted with a disordermediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman'sdisease and AIDS-associated primary effusion lymphoma. In someembodiments, the expression of a KSHV gene is reduced. In someembodiments the expression of a human gene that is required for KSHVreplication is reduced.

The invention also includes a method for treating patients infected bythe JC Virus (JCV) or a disease or disorder associated with this virus,e.g., progressive multifocal leukoencephalopathy (PML). In someembodiments, the expression of a JCV gene is reduced. In certainembodiments the expression of a human gene that is required for JCVreplication is reduced.

Methods of the invention also provide for treating patients infected bythe myxovirus or at risk for or afflicted with a disorder mediated bymyxovirus, e.g., influenza. In some embodiments, the expression of amyxovirus gene is reduced. In some embodiments the expression of a humangene that is required for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe rhinovirus or at risk for of afflicted with a disorder mediated byrhinovirus, e.g., the common cold. In some embodiments, the expressionof a rhinovirus gene is reduced. In certain embodiments the expressionof a human gene that is required for rhinovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe coronavirus or at risk for of afflicted with a disorder mediated bycoronavirus, e.g., the common cold. In some embodiments, the expressionof a coronavirus gene is reduced. In certain embodiments the expressionof a human gene that is required for coronavirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe flavivirus West Nile or at risk for or afflicted with a disordermediated by West Nile Virus. In some embodiments, the expression of aWest Nile Virus gene is reduced. In another embodiment, the West NileVirus gene is one of the group comprising E, NS3, or NS5. In someembodiments the expression of a human gene that is required for WestNile Virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe St. Louis Encephalitis flavivirus, or at risk for or afflicted witha disease or disorder associated with this virus, e.g., viralhaemorrhagic fever or neurological disease. In some embodiments, theexpression of a St. Louis Encephalitis gene is reduced. In someembodiments the expression of a human gene that is required for St.Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe Tick-borne encephalitis flavivirus, or at risk for or afflicted witha disorder mediated by Tick-borne encephalitis virus, e.g., viralhaemorrhagic fever and neurological disease. In some embodiments, theexpression of a Tick-borne encephalitis virus gene is reduced. In someembodiments the expression of a human gene that is required forTick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patientsinfected by the Murray Valley encephalitis flavivirus, which commonlyresults in viral haemorrhagic fever and neurological disease. In someembodiments, the expression of a Murray Valley encephalitis virus geneis reduced. In some embodiments the expression of a human gene that isrequired for Murray Valley encephalitis virus replication is reduced.

The invention also includes methods for treating patients infected bythe dengue flavivirus, or a disease or disorder associated with thisvirus, e.g., dengue haemorrhagic fever. In some embodiments, theexpression of a dengue virus gene is reduced. In some embodiments theexpression of a human gene that is required for dengue virus replicationis reduced.

Methods of the invention also provide for treating patients infected bythe Simian Virus 40 (SV40) or at risk for or afflicted with a disordermediated by SV40, e.g., tumorigenesis. In some embodiments, theexpression of a SV40 gene is reduced. In some embodiments the expressionof a human gene that is required for SV40 replication is reduced.

The invention also includes methods for treating patients infected bythe Human T Cell Lymphotropic Virus (HTLV), or a disease or disorderassociated with this virus, e.g., leukemia and myelopathy. In someembodiments, the expression of a HTLV gene is reduced. In anotherembodiment the HTLV1 gene is the Tax transcriptional activator. In someembodiments the expression of a human gene that is required for HTLVreplication is reduced.

Methods of the invention also provide for treating patients infected bythe Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflictedwith a disorder mediated by Mo-MuLV, e.g., T-cell leukemia. In someembodiments, the expression of a Mo-MuLV gene is reduced. In someembodiments the expression of a human gene that is required for Mo-MuLVreplication is reduced.

Methods of the invention also provide for treating patients infected bythe encephalomyocarditis virus (EMCV) or at risk for or afflicted with adisorder mediated by EMCV, e.g., myocarditis. EMCV leads to myocarditisin mice and pigs and is capable of infecting human myocardial cells.This virus is therefore a concern for patients undergoingxenotransplantation. In some embodiments, the expression of a EMCV geneis reduced. In some embodiments the expression of a human gene that isrequired for EMCV replication is reduced.

The invention also includes a method for treating patients infected bythe measles virus (MV) or at risk for or afflicted with a disordermediated by MV, e.g., measles. In some embodiments, the expression of aMV gene is reduced. In some embodiments the expression of a human genethat is required for MV replication is reduced.

The invention also includes a method for treating patients infected bythe Vericella zoster virus (VZV) or at risk for or afflicted with adisorder mediated by VZV, e.g., chicken pox or shingles (also calledzoster). In some embodiments, the expression of a VZV gene is reduced.In some embodiments the expression of a human gene that is required forVZV replication is reduced.

The invention also includes a method for treating patients infected byan adenovirus or at risk for or afflicted with a disorder mediated by anadenovirus, e.g., respiratory tract infection. In some embodiments, theexpression of an adenovirus gene is reduced. In some embodiments theexpression of a human gene that is required for adenovirus replicationis reduced.

The invention includes a method for treating patients infected by ayellow fever virus (YFV) or at risk for or afflicted with a disordermediated by a YFV, e.g., respiratory tract infection. In someembodiments, the expression of a YFV gene is reduced. In anotherembodiment, the gene may be one of a group that includes the E, NS2A, orNS3 genes. In some embodiments the expression of a human gene that isrequired for YFV replication is reduced.

Methods of the invention also provide for treating patients infected bythe poliovirus or at risk for or afflicted with a disorder mediated bypoliovirus, e.g., polio. In some embodiments, the expression of apoliovirus gene is reduced. In some embodiments the expression of ahuman gene that is required for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected bya poxvirus or at risk for or afflicted with a disorder mediated by apoxvirus, e.g., smallpox. In some embodiments, the expression of apoxvirus gene is reduced. In some embodiments the expression of a humangene that is required for poxvirus replication is reduced.

Other Pathogens

In another, aspect the invention features methods of treating a subjectinfected with a pathogen, e.g., a bacterial, amoebic, parasitic, orfungal pathogen. The method comprises administering an iRNA of theinvention to a subject in need thereof, thereby treating the subject.The iRNA agent that is administered will depend on the type of pathogenbeing treated. In some embodiments, the iRNA agent may target a pathogengene. In other embodiments, the nucleic acid may target a host gene.

The target gene can be one involved in growth, cell wall synthesis,protein synthesis, transcription, energy metabolism, e.g., the Krebscycle, or toxin production.

Thus, the present invention provides for a method of treating patientsinfected by a plasmodium that causes malaria. In some embodiments, theexpression of a plasmodium gene is reduced. In another embodiment, thegene is apical membrane antigen 1 (AMA1). In some embodiments theexpression of a human gene that is required for plasmodium replicationis reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium ulcerans, or a disease or disorder associated withthis pathogen, e.g., Buruli ulcers. In some embodiments, the expressionof a Mycobacterium ulcerans gene is reduced. In some embodiments theexpression of a human gene that is required for Mycobacterium ulceransreplication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium tuberculosis, or a disease or disorder associated withthis pathogen, e.g., tuberculosis. In some embodiments, the expressionof a Mycobacterium tuberculosis gene is reduced. In some embodiments theexpression of a human gene that is required for Mycobacteriumtuberculosis replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium leprae, or a disease or disorder associated with thispathogen, e.g., leprosy. In some embodiments, the expression of aMycobacterium leprae gene is reduced. In some embodiments the expressionof a human gene that is required for Mycobacterium leprae replication isreduced.

The invention also includes methods for treating patients infected bythe bacteria Staphylococcus aureus, or a disease or disorder associatedwith this pathogen, e.g., infections of the skin and muscous membranes.In some embodiments, the expression of a Staphylococcus aureus gene isreduced. In some embodiments the expression of a human gene that isrequired for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pneumoniae, or a disease or disorderassociated with this pathogen, e.g., pneumonia or childhood lowerrespiratory tract infection. In some embodiments, the expression of aStreptococcus pneumoniae gene is reduced. In some embodiments theexpression of a human gene that is required for Streptococcus pneumoniaereplication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pyogenes, or a disease or disorder associatedwith this pathogen, e.g., Strep throat or Scarlet fever. In someembodiments, the expression of a Streptococcus pyogenes gene is reduced.In some embodiments the expression of a human gene that is required forStreptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Chlamydia pneumoniae, or a disease or disorder associatedwith this pathogen, e.g., pneumonia or childhood lower respiratory tractinfection. In some embodiments, the expression of a Chlamydia pneumoniaegene is reduced. In some embodiments the expression of a human gene thatis required for Chlamydia pneumoniae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Mycoplasma pneumoniae, or a disease or disorder associatedwith this pathogen, e.g., pneumonia or childhood lower respiratory tractinfection. In some embodiments, the expression of a Mycoplasmapneumoniae gene is reduced. In some embodiments the expression of ahuman gene that is required for Mycoplasma pneumoniae replication isreduced.

Immune Disorders

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with a disease or disordercharacterized by an unwanted immune response, e.g., an inflammatorydisease or disorder, or an autoimmune disease or disorder. The methodcomprises administering an iRNA agent of the invention to a subject inneed thereof, thereby treating the subject. The iRNA agent that isadministered will depend on the type of immune disorder being treated.

In some embodiments the disease or disorder is an ischemia orreperfusion injury, e.g., ischemia or reperfusion injury associated withacute myocardial infarction, unstable angina, cardiopulmonary bypass,surgical intervention e.g., angioplasty, e.g., percutaneous transluminalcoronary angioplasty, the response to a transplantated organ or tissue,e.g., transplanted cardiac or vascular tissue; or thrombolysis.

In some embodiments the disease or disorder is restenosis, e.g.,restenosis associated with surgical intervention e.g., angioplasty,e.g., percutaneous transluminal coronary angioplasty.

In certain embodiments the disease or disorder is Inflammatory BowelDisease, e.g., Crohn Disease or Ulcerative Colitis.

In certain embodiments the disease or disorder is inflammationassociated with an infection or injury.

In certain embodiments the disease or disorder is asthma, lupus,multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g.,rheumatoid or psoriatic.

In certain other embodiments the iRNA agent silences an integrin orco-ligand thereof, e.g., VLA4, VCAM, ICAM.

In certain other embodiments the iRNA agent silences a selectin orco-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin,P-selectin glycoprotein-1 (PSGL-1).

In certain other embodiments the iRNA agent silences a component of thecomplement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5convertase.

In certain other embodiments the iRNA agent silences a chemokine orreceptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2, IL-2R, IL-4,IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.

In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3,PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES,MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, 1-309.

Pain

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with acute pain or chronic pain.The method comprises administering an iRNA agent of the invention to asubject in need thereof, thereby treating the subject. The iRNA agentthat is administered will depend on the type of pain being treated.

In certain other embodiments the iRNA agent silences a component of anion channel.

In certain other embodiments the iRNA agent silences a neurotransmitterreceptor or ligand.

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with a neurological disease ordisorder. The method includes:

providing an iRNA agent which iRNA is homologous to and can silence,e.g., by cleavage, a gene which mediates a neurological disease ordisorder;

administering the to a subject,

thereby treating the subject.

Neurological Disorders

In certain embodiments the disease or disorder is a neurologicaldisorder, including Alzheimer's Disease or Parkinson Disease. The methodcomprises administering an iRNA agent of the invention to a subject inneed thereof, thereby treating the subject. The iRNA agent that isadministered will depend on the type of neurological disorder beingtreated.

In certain other embodiments the iRNA agent silences an amyloid-familygene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, orI-synuclein.

In some embodiments the disease or disorder is a neurodegenerativetrinucleotide repeat disorder, e.g., Huntington disease, dentatorubralpallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2,SCA3 (Machado-Joseph disease), SCA7 or SCA8.

In certain other embodiments the iRNA agent silences HD, DRPLA, SCA1,SCA2, MJD1, CACNL1A4, SCA7, SCA8.

Loss of Heterozygosity

The loss of heterozygosity (LOH) can result in hemizygosity forsequence, e.g., genes, in the area of LOH. This can result in asignificant genetic difference between normal and disease-state cells,e.g., cancer cells, and provides a useful difference between normal anddisease-state cells, e.g., cancer cells. This difference can arisebecause a gene or other sequence is heterozygous in euploid cells but ishemizygous in cells having LOH. The regions of LOH will often include agene, the loss of which promotes unwanted proliferation, e.g., a tumorsuppressor gene, and other sequences including, e.g., other genes, insome cases a gene which is essential for normal function, e.g., growth.Methods of the invention rely, in part, on the specific cleavage orsilencing of one allele of an essential gene with an iRNA agent of theinvention. The iRNA agent is selected such that it targets the singleallele of the essential gene found in the cells having LOH but does notsilence the other allele, which is present in cells which do not showLOH. In essence, it discriminates between the two alleles,preferentially silencing the selected allele. In essence polymorphisms,e.g., SNPs of essential genes that are affected by LOH, are used as atarget for a disorder characterized by cells having LOH, e.g., cancercells having LOH.

One of ordinary skill in the art can identify essential genes which arein proximity to tumor suppressor genes, and which are within a LOHregion which includes the tumor suppressor gene. The gene encoding thelarge subunit of human RNA polymerase II, POLR2A, a gene located inclose proximity to the tumor suppressor gene p53, is such a gene. Itfrequently occurs within a region of LOH in cancer cells. Other genesthat occur within LOH regions and are lost in many cancer cell typesinclude the group comprising replication protein A 70-kDa subunit,replication protein A 32-kD, ribonucleotide reductase, thymidilatesynthase, TATA associated factor 2H, ribosomal protein S14, eukaryoticinitiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase,NaK ATPase, alpha-1 subunit, and transferrin receptor.

Accordingly, the invention features, a method of treating a disordercharacterized by LOH, e.g., cancer. The method comprises optionally,determining the genotype of the allele of a gene in the region of LOHand determining the genotype of both alleles of the gene in a normalcell; providing an iRNA agent which preferentially cleaves or silencesthe allele found in the LOH cells; and administering the iRNA to thesubject, thereby treating the disorder.

The invention also includes a iRNA agent disclosed herein, e.g, an iRNAagent which can preferentially silence, e.g., cleave, one allele of apolymorphic gene.

In another aspect, the invention provides a method of cleaving orsilencing more than one gene with an iRNA agent. In these embodimentsthe iRNA agent is selected so that it has sufficient homology to asequence found in more than one gene. For example, the sequenceAAGCTGGCCCTGGACATGGAGAT (SEQ ID NO: 22) is conserved between mouse laminB1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agenttargeted to this sequence would effectively silence the entirecollection of genes.

The invention also includes an iRNA agent disclosed herein, which cansilence more than one gene.

Routes of Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It may be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Acomposition that includes a iRNA can be delivered to a subject by avariety of routes. Exemplary routes include: intravenous, topical,rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of iRNA and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, elixirs or non-aqueous media,tablets, capsules, lozenges, or troches. In the case of tablets,carriers that can be used include lactose, sodium citrate and salts ofphosphoric acid. Various disintegrants such as starch, and lubricatingagents such as magnesium stearate, sodium lauryl sulfate and talc, arecommonly used in tablets. For oral administration in capsule form,useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous suspensions are required for oral use, the nucleicacid compositions can be combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents can beadded.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes may be controlled torender the preparation isotonic.

For ocular administration, ointments or droppable liquids may bedelivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It may be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. In someembodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) is delivered to a subject via topical administration. “Topicaladministration” refers to the delivery to a subject by contacting theformulation directly to a surface of the subject. The most common formof topical delivery is to the skin, but a composition disclosed hereincan also be directly applied to other surfaces of the body, e.g., to theeye, a mucous membrane, to surfaces of a body cavity or to an internalsurface.

As mentioned above, the most common topical delivery is to the skin. Theterm encompasses several routes of administration including, but notlimited to, topical and transdermal. These modes of administrationtypically include penetration of the skin's permeability barrier andefficient delivery to the target tissue or stratum. Topicaladministration can be used as a means to penetrate the epidermis anddermis and ultimately achieve systemic delivery of the composition.Topical administration can also be used as a means to selectivelydeliver oligonucleotides to the epidermis or dermis of a subject, or tospecific strata thereof, or to an underlying tissue.

The term “skin,” as used herein, refers to the epidermis and/or dermisof an animal. Mammalian skin consists of two major, distinct layers. Theouter layer of the skin is called the epidermis. The epidermis iscomprised of the stratum corneum, the stratum granulosum, the stratumspinosum, and the stratum basale, with the stratum comeum being at thesurface of the skin and the stratum basale being the deepest portion ofthe epidermis. The epidermis is between 50 μm and 0.2 mm thick,depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker thanthe epidermis. The dermis is primarily composed of collagen in the formof fibrous bundles. The collagenous bundles provide support for, interalia, blood vessels, lymph capillaries, glands, nerve endings andimmunologically active cells.

One of the major functions of the skin as an organ is to regulate theentry of substances into the body. The principal permeability barrier ofthe skin is provided by the stratum comeum, which is formed from manylayers of cells in various states of differentiation. The spaces betweencells in the stratum comeum is filled with different lipids arranged inlattice-like formations that provide seals to further enhance the skinspermeability barrier.

The permeability barrier provided by the skin is such that it is largelyimpermeable to molecules having molecular weight greater than about 750Da. For larger molecules to cross the skin's permeability barrier,mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administeredagents. These factors include the characteristics of the treated skin,the characteristics of the delivery agent, interactions between both thedrug and delivery agent and the drug and skin, the dosage of the drugapplied, the form of treatment, and the post treatment regimen. Toselectively target the epidermis and dermis, it is sometimes possible toformulate a composition that comprises one or more penetration enhancersthat will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163),phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 166), and optimization ofvehicle characteristics relative to dose position and retention at thesite of administration (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 168) may be useful methods for enhancing thetransport of topically applied compositions across skin and mucosalsites.

The compositions and methods provided may also be used to examine thefunction of various proteins and genes in vitro in cultured or preserveddermal tissues and in animals. The invention can be thus applied toexamine the function of any gene. The methods of the invention can alsobe used therapeutically or prophylactically. For example, for thetreatment of animals that are known or suspected to suffer from diseasessuch as psoriasis, lichen planus, toxic epidermal necrolysis, ertythemamultiforme, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lymedisease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It may be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Acomposition that includes an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof) can be administered to a subject by pulmonarydelivery. Pulmonary delivery compositions can be delivered by inhalationby the patient of a dispersion so that the composition, for example,iRNA, within the dispersion can reach the lung where it can be readilyabsorbed through the alveolar region directly into blood circulation.Pulmonary delivery can be effective both for systemic delivery and forlocalized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are may be used. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. A iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” For example, theaverage particle size is less than about 10 μm in diameter with arelatively uniform spheroidal shape distribution. In some embodiments,the diameter is less than about 7.5 μm and in some embodiments less thanabout 5.0 μm. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, sometimes about 0.3 μm to about 5 μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and in somecases less it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A group of carbohydrates may includeslactose, threhalose, raffinose maltodextrins, and mannitol. Suitablepolypeptides include aspartame. Amino acids include alanine and glycine,with glycine being used in some embodiments.

Additives, which are minor components of the composition of thisinvention, may be included for conformational stability during spraydrying and for improving dispersibility of the powder. These additivesinclude hydrophobic amino acids such as tryptophan, tyrosine, leucine,phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate may be used in some embodiments.

Pulmonary administration of a micellar iRNA formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Oral or Nasal Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It may be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Boththe oral and nasal membranes offer advantages over other routes ofadministration. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many drugs. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears tobe related to molecular size, lipid solubility and peptide proteinionization. Small molecules, less than 1000 daltons appear to crossmucosa rapidly. As molecular size increases, the permeability decreasesrapidly. Lipid soluble compounds are more permeable than non-lipidsoluble molecules. Maximum absorption occurs when molecules areun-ionized or neutral in electrical charges. Therefore charged moleculespresent the biggest challenges to absorption through the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to thebuccal cavity of a human being by spraying into the cavity, withoutinhalation, from a metered dose spray dispenser, a mixed micellarpharmaceutical formulation as described above and a propellant. In oneembodiment, the dispenser is first shaken prior to spraying thepharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It may be understood, however, that these devices,formulations, compositions and methods can be practiced with other iRNAagents, e.g., modified iRNA agents, and such practice is within theinvention. An iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) can be disposed on or in a device, e.g., a device whichimplanted or otherwise placed in a subject. Exemplary devices includedevices which are introduced into the vasculature, e.g., devicesinserted into the lumen of a vascular tissue, or which devicesthemselves form a part of the vasculature, including stents, catheters,heart valves, and other vascular devices. These devices, e.g., cathetersor stents, can be placed in the vasculature of the lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to aiRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and otherorthopedic implants.

In one embodiment, unit doses or measured doses of a composition thatincludes iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include pump, e.g., and, optionally, associatedelectronics.

Tissue, e.g., cells or organs can be treated with An iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof) ex vivo and then administered orimplanted in a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g.,tissue can be treated to reduce graft v. host disease. In otherembodiments, the tissue is allogeneic and the tissue is treated to treata disorder characterized by unwanted gene expression in that tissue.E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoieticcells, can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can becombined with other therapies.

In some implementations, the iRNA treated cells are insulated from othercells, e.g., by a semi-permeable porous barrier that prevents the cellsfrom leaving the implant, but enables molecules from the body to reachthe cells and molecules produced by the cells to enter the body. In oneembodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a siRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, or precursor thereof). Exemplary devicesinclude condoms, diaphragms, IUD (implantable uterine devices, sponges,vaginal sheaths, and birth control devices. In one embodiment, the iRNAis chosen to inactive sperm or egg. In another embodiment, the iRNA ischosen to be complementary to a viral or pathogen RNA, e.g., an RNA ofan STD. In some instances, the iRNA composition can include aspermicide.

Dosage

In one aspect, the invention features a method of administering an iRNAagent, e.g., a double-stranded iRNA agent, or siRNA agent, to a subject(e.g., a human subject). The method includes administering a unit doseof the iRNA agent, e.g., a siRNA agent, e.g., double stranded siRNAagent that (a) the double-stranded part is 19-25 nucleotides (nt) long,for example, 21-23 nt, (b) is complementary to a target RNA (e.g., anendogenous or pathogen target RNA), and, optionally, (c) includes atleast one 3′ overhang 1-5 nucleotide long. In one embodiment, the unitdose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1,0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g.,about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750,300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015,0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with thetarget RNA. The unit dose, for example, can be administered by injection(e.g., intravenous or intramuscular), an inhaled dose, or a topicalapplication. In some embodiments dosages may be less than 2, 1, or 0.1mg/kg of body weight.

In some embodiments, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject has aviral infection and the modality is an antiviral agent other than aniRNA agent, e.g., other than a double-stranded iRNA agent, or siRNAagent. In another embodiment, the subject has atherosclerosis and theeffective dose of an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, is administered in combination with, e.g., after surgicalintervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g.,10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. Themaintenance doses are, for example, administered no more than once every5, 10, or 30 days. Further, the treatment regimen may last for a periodof time which will vary depending upon the nature of the particulardisease, its severity and the overall condition of the patient. Incertain embodiments the dosage may be delivered no more than once perday, e.g., no more than once per 24, 36, 48, or more hours, e.g., nomore than once for every 5 or 8 days. Following treatment, the patientcan be monitored for changes in his condition and for alleviation of thesymptoms of the disease state. The dosage of the compound may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disease state is observed, if the disease state hasbeen ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. In another embodiment, the iRNA agentspecies has sequences that are non-overlapping and non-adjacent toanother species with respect to a naturally occurring target sequence.In another embodiment, the plurality of iRNA agent species is specificfor different naturally occurring target genes. In another embodiment,the iRNA agent is allele specific.

In some cases, a patient is treated with a iRNA agent in conjunctionwith other therapeutic modalities. For example, a patient being treatedfor a viral disease, e.g., an HIV associated disease (e.g., AIDS), maybe administered a iRNA agent specific for a target gene essential to thevirus in conjunction with a known antiviral agent (e.g., a proteaseinhibitor or reverse transcriptase inhibitor). In another example, apatient being treated for cancer may be administered a iRNA agentspecific for a target essential for tumor cell proliferation inconjunction with a chemotherapy.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 g to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g., nasal, buccal, pulmonary.For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof) can include a single treatment or,for example, can include a series of treatments. It will also beappreciated that the effective dosage of a iRNA agent such as a siRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays as described herein. For example,the subject can be monitored after administering a iRNA agentcomposition. Based on information from the monitoring, an additionalamount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA. The transgenic animal can be deficient for thecorresponding endogenous RNA. In another embodiment, the composition fortesting includes a iRNA agent that is complementary, at least in aninternal region, to a sequence that is conserved between the target RNAin the animal model and the target RNA in a human.

The inventors have discovered that iRNA agents described herein can beadministered to mammals, particularly large mammals such as nonhumanprimates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, composition is parenteral,e.g., intravenous (e.g., as a bolus or as a diffusible infusion),intradermal, intraperitoneal, intramuscular, intrathecal,intraventricular, intracranial, subcutaneous, transmucosal, buccal,sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary,intranasal, urethral or ocular. Administration can be provided by thesubject or by another person, e.g., a health care provider. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose. Selected modes of delivery are discussed inmore detail below.

The invention provides methods, compositions, and kits, for rectaladministration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes a an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) described herein, e.g., a therapeutically effective amount of aiRNA agent described herein, e.g., a iRNA agent having a double strandedregion of less than 40, and, for example, less than 30 nucleotides andhaving one or two 1-3 nucleotide single strand 3′ overhangs can beadministered rectally, e.g., introduced through the rectum into thelower or upper colon. This approach is particularly useful in thetreatment of, inflammatory disorders, disorders characterized byunwanted cell proliferation, e.g., polyps, or colon cancer.

The medication can be delivered to a site in the colon by introducing adispensing device, e.g., a flexible, camera-guided device similar tothat used for inspection of the colon or removal of polyps, whichincludes means for delivery of the medication.

The rectal administration of the iRNA agent is by means of an enema. TheiRNA agent of the enema can be dissolved in a saline or bufferedsolution. The rectal administration can also by means of a suppository,which can include other ingredients, e.g., an excipient, e.g., cocoabutter or hydropropylmethylcellulose.

Any of the iRNA agents described herein can be administered orally,e.g., in the form of tablets, capsules, gel capsules, lozenges, trochesor liquid syrups. Further, the composition can be applied topically to asurface of the oral cavity.

Any of the iRNA agents described herein can be administered buccally.For example, the medication can be sprayed into the buccal cavity orapplied directly, e.g., in a liquid, solid, or gel form to a surface inthe buccal cavity. This administration is particularly desirable for thetreatment of inflammations of the buccal cavity, e.g., the gums ortongue, e.g., in one embodiment, the buccal administration is byspraying into the cavity, e.g., without inhalation, from a dispenser,e.g., a metered dose spray dispenser that dispenses the pharmaceuticalcomposition and a propellant.

Any of the iRNA agents described herein can be administered to oculartissue. For example, the medications can be applied to the surface ofthe eye or nearby tissue, e.g., the inside of the eyelid. They can beapplied topically, e.g., by spraying, in drops, as an eyewash, or anointment. Administration can be provided by the subject or by anotherperson, e.g., a health care provider. The medication can be provided inmeasured doses or in a dispenser which delivers a metered dose. Themedication can also be administered to the interior of the eye, and canbe introduced by a needle or other delivery device which can introduceit to a selected area or structure. Ocular treatment is particularlydesirable for treating inflammation of the eye or nearby tissue.

Any of the iRNA agents described herein can be administered directly tothe skin. For example, the medication can be applied topically ordelivered in a layer of the skin, e.g., by the use of a microneedle or abattery of microneedles which penetrate into the skin, but, for example,not into the underlying muscle tissue. Administration of the iRNA agentcomposition can be topical. Topical applications can, for example,deliver the composition to the dermis or epidermis of a subject. Topicaladministration can be in the form of transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids or powders.A composition for topical administration can be formulated as aliposome, micelle, emulsion, or other lipophilic molecular assembly. Thetransdermal administration can be applied with at least one penetrationenhancer, such as iontophoresis, phonophoresis, and sonophoresis.

Any of the iRNA agents described herein can be administered to thepulmonary system. Pulmonary administration can be achieved by inhalationor by the introduction of a delivery device into the pulmonary system,e.g., by introducing a delivery device which can dispense themedication. Certain embodiments may use a method of pulmonary deliveryby inhalation. The medication can be provided in a dispenser whichdelivers the medication, e.g., wet or dry, in a form sufficiently smallsuch that it can be inhaled. The device can deliver a metered dose ofmedication. The subject, or another person, can administer themedication.

Pulmonary delivery is effective not only for disorders which directlyaffect pulmonary tissue, but also for disorders which affect othertissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or aerosol for pulmonary delivery.

Any of the iRNA agents described herein can be administered nasally.Nasal administration can be achieved by introduction of a deliverydevice into the nose, e.g., by introducing a delivery device which candispense the medication. Methods of nasal delivery include spray,aerosol, liquid, e.g., by drops, or by topical administration to asurface of the nasal cavity. The medication can be provided in adispenser with delivery of the medication, e.g., wet or dry, in a formsufficiently small such that it can be inhaled. The device can deliver ametered dose of medication. The subject, or another person, canadminister the medication.

Nasal delivery is effective not only for disorders which directly affectnasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or for nasal delivery.

An iRNA agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

The dosage of a pharmaceutical composition including a iRNA agent can beadministered in order to alleviate the symptoms of a disease state,e.g., cancer or a cardiovascular disease. A subject can be treated withthe pharmaceutical composition by any of the methods mentioned above.

Gene expression in a subject can be modulated by administering apharmaceutical composition including an iRNA agent.

A subject can be treated by administering a defined amount of an iRNAagent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a siRNAagent) composition that is in a powdered form, e.g., a collection ofmicroparticles, such as crystalline particles. The composition caninclude a plurality of iRNA agents, e.g., specific for one or moredifferent endogenous target RNAs. The method can include other featuresdescribed herein.

A subject can be treated by administering a defined amount of an iRNAagent composition that is prepared by a method that includesspray-drying, i.e., atomizing a liquid solution, emulsion, orsuspension, immediately exposing the droplets to a drying gas, andcollecting the resulting porous powder particles. The composition caninclude a plurality of iRNA agents, e.g., specific for one or moredifferent endogenous target RNAs. The method can include other featuresdescribed herein.

The iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent,(e.g., a precursor, e.g., a larger iRNA agent which can be processedinto a siRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, or precursor thereof), canbe provided in a powdered, crystallized or other finely divided form,with or without a carrier, e.g., a micro- or nano-particle suitable forinhalation or other pulmonary delivery. This can include providing anaerosol preparation, e.g., an aerosolized spray-dried composition. Theaerosol composition can be provided in and/or dispensed by a metereddose delivery device.

The subject can be treated for a condition treatable by inhalation,e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNAagent which can be processed into a siRNA agent, or a DNA which encodesan iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof) composition and inhaling the aerosolized composition.The iRNA agent can be an siRNA. The composition can include a pluralityof iRNA agents, e.g., specific for one or more different endogenoustarget RNAs. The method can include other features described herein.

A subject can be treated by, for example, administering a compositionincluding an effective/defined amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof), wherein the composition is preparedby a method that includes spray-drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques.

In another aspect, the invention features a method that includes:evaluating a parameter related to the abundance of a transcript in acell of a subject; comparing the evaluated parameter to a referencevalue; and if the evaluated parameter has a preselected relationship tothe reference value (e.g., it is greater), administering a iRNA agent(or a precursor, e.g., a larger iRNA agent which can be processed into asiRNA agent, or a DNA which encodes a iRNA agent or precursor thereof)to the subject. In one embodiment, the iRNA agent includes a sequencethat is complementary to the evaluated transcript. For example, theparameter can be a direct measure of transcript levels, a measure of aprotein level, a disease or disorder symptom or characterization (e.g.,rate of cell proliferation and/or tumor mass, viral load).

In another aspect, the invention features a method that includes:administering a first amount of a composition that comprises an iRNAagent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a siRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, or precursor thereof) to a subject, whereinthe iRNA agent includes a strand substantially complementary to a targetnucleic acid; evaluating an activity associated with a protein encodedby the target nucleic acid; wherein the evaluation is used to determineif a second amount may be administered. In some embodiments the methodincludes administering a second amount of the composition, wherein thetiming of administration or dosage of the second amount is a function ofthe evaluating. The method can include other features described herein.

In another aspect, the invention features a method of administering asource of a double-stranded iRNA agent (ds iRNA agent) to a subject. Themethod includes administering or implanting a source of a ds iRNA agent,e.g., a siRNA agent, that (a) includes a double-stranded region that is19-25 nucleotides long, for example, 21-23 nucleotides, (b) iscomplementary to a target RNA (e.g., an endogenous RNA or a pathogenRNA), and, optionally, (c) includes at least one 3′ overhang 1-5 ntlong. In one embodiment, the source releases ds iRNA agent over time,e.g., the source is a controlled or a slow release source, e.g., amicroparticle that gradually releases the ds iRNA agent. In anotherembodiment, the source is a pump, e.g., a pump that includes a sensor ora pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) including a nucleotide sequence complementary to a target RNA,e.g., substantially and/or exactly complementary. The target RNA can bea transcript of an endogenous human gene. In one embodiment, the iRNAagent (a) is 19-25 nucleotides long, for example, 21-23 nucleotides, (b)is complementary to an endogenous target RNA, and, optionally, (c)includes at least one 3′ overhang 1-5 nt long. In one embodiment, thepharmaceutical composition can be an emulsion, microemulsion, cream,jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agentmixed with a topical delivery agent. The topical delivery agent can be aplurality of microscopic vesicles. The microscopic vesicles can beliposomes. In some embodiments the liposomes are cationic liposomes.

In another aspect, the pharmaceutical composition includes an iRNAagent, e.g., a double-stranded iRNA agent, or siRNA agent (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a siRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, or precursor thereof) admixed with a topicalpenetration enhancer. In one embodiment, the topical penetrationenhancer is a fatty acid. The fatty acid can be arachidonic acid, oleicacid, lauric acid, caprylic acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or aC₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt.The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid,glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid,sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable saltthereof.

In another embodiment, the penetration enhancer is a chelating agent.The chelating agent can be EDTA, citric acid, a salicyclate, a N-acylderivative of collagen, laureth-9, an N-amino acyl derivative of abeta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g.,an ionic or nonionic surfactant. The surfactant can be sodium laurylsulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether,a perfluorchemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from agroup consisting of unsaturated cyclic ureas, 1-alkyl-alkones,1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents andmixtures thereof. In yet another embodiment the penetration enhancer canbe a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) in a form suitable for oral delivery. In one embodiment, oraldelivery can be used to deliver an iRNA agent composition to a cell or aregion of the gastrointestinal tract, e.g., small intestine, colon(e.g., to treat a colon cancer), and so forth. The oral delivery formcan be tablets, capsules or gel capsules. In one embodiment, the iRNAagent of the pharmaceutical composition modulates expression of acellular adhesion protein, modulates a rate of cellular proliferation,or has biological activity against eukaryotic pathogens or retroviruses.In another embodiment, the pharmaceutical composition includes anenteric material that substantially prevents dissolution of the tablets,capsules or gel capsules in a mammalian stomach. In some embodiments theenteric material is a coating. The coating can be acetate phthalate,propylene glycol, sorbitan monoleate, cellulose acetate trimellitate,hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a penetration enhancer. The penetration enhancercan be a bile salt or a fatty acid. The bile salt can be ursodeoxycholicacid, chenodeoxycholic acid, and salts thereof. The fatty acid can becapric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes an excipient. In one example the excipient ispolyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a plasticizer. The plasticizer can be diethylphthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethylcitrate.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent and a delivery vehicle. In one embodiment, theiRNA agent is (a) is 19-25 nucleotides long, for example, 21-23nucleotides, (b) is complementary to an endogenous target RNA, and,optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g.,a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g.,a larger iRNA agent which can be processed into a siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof) to a cell by a topical route ofadministration. The delivery vehicle can be microscopic vesicles. In oneexample the microscopic vesicles are liposomes. In some embodiments theliposomes are cationic liposomes. In another example the microscopicvesicles are micelles. In one aspect, the invention features apharmaceutical composition including an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof) in an injectable dosage form. In oneembodiment, the injectable dosage form of the pharmaceutical compositionincludes sterile aqueous solutions or dispersions and sterile powders.In some embodiments the sterile solution can include a diluent such aswater; saline solution; fixed oils, polyethylene glycols, glycerin, orpropylene glycol.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) in oral dosage form. In one embodiment, the oral dosage form isselected from the group consisting of tablets, capsules and gelcapsules. In another embodiment, the pharmaceutical composition includesan enteric material that substantially prevents dissolution of thetablets, capsules or gel capsules in a mammalian stomach. In someembodiments the enteric material is a coating. The coating can beacetate phthalate, propylene glycol, sorbitan monoleate, celluloseacetate trimellitate, hydroxy propyl methyl cellulose phthalate orcellulose acetate phthalate. In one embodiment, the oral dosage form ofthe pharmaceutical composition includes a penetration enhancer, e.g., apenetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes an excipient. In one example the excipient ispolyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a plasticizer. The plasticizer can be diethylphthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethylcitrate.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) in a rectal dosage form. In one embodiment, the rectal dosageform is an enema. In another embodiment, the rectal dosage form is asuppository.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) in a vaginal dosage form. In one embodiment, the vaginal dosageform is a suppository. In another embodiment, the vaginal dosage form isa foam, cream, or gel.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) in a pulmonary or nasal dosage form. In one embodiment, theiRNA agent is incorporated into a particle, e.g., a macroparticle, e.g.,a microsphere. The particle can be produced by spray drying,lyophilization, evaporation, fluid bed drying, vacuum drying, or acombination thereof. The microsphere can be formulated as a suspension,a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof) composition suitable for inhalationby a subject, including: (a) a therapeutically effective amount of aiRNA agent suitable for treating a condition in the subject byinhalation; (b) a pharmaceutically acceptable excipient selected fromthe group consisting of carbohydrates and amino acids; and (c)optionally, a dispersibility-enhancing amount of aphysiologically-acceptable, water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate canbe selected from the group consisting of monosaccharides, disaccharides,trisaccharides, and polysaccharides. In some embodiments thecarbohydrate is a monosaccharide selected from the group consisting ofdextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. Inanother embodiment the carbohydrate is a disaccharide selected from thegroup consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In oneembodiment, the amino acid is a hydrophobic amino acid. In someembodiments the hydrophobic amino acid is selected from the groupconsisting of alanine, isoleucine, leucine, methionine, phenylalanine,proline, tryptophan, and valine. In yet another embodiment the aminoacid is a polar amino acid. In some embodiments the amino acid isselected from the group consisting of arginine, histidine, lysine,cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acidand glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selectedfrom the group consisting of human serum albumin, α-lactalbumin,trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter (MMD) of less than 10 microns.In another embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter of less than 5 microns. In yetanother embodiment the spray-dried iRNA agent composition includesparticles having a mass median aerodynamic diameter (MMAD) of less than5 microns.

In certain other aspects, the invention provides kits that include asuitable container containing a pharmaceutical formulation of an iRNAagent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a siRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or siRNA agent, or precursor thereof). In certainembodiments the individual components of the pharmaceutical formulationmay be provided in one container. Alternatively, it may be desirable toprovide the components of the pharmaceutical formulation separately intwo or more containers, e.g., one container for an iRNA agentpreparation, and at least another for a carrier compound. The kit may bepackaged in a number of different configurations such as one or morecontainers in a single box. The different components can be combined,e.g., according to instructions provided with the kit. The componentscan be combined according to a method described herein, e.g., to prepareand administer a pharmaceutical composition. The kit can also include adelivery device.

In another aspect, the invention features a device, e.g., an implantabledevice, wherein the device can dispense or administer a composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof), e.g., a iRNA agent that silences an endogenous transcript. Inone embodiment, the device is coated with the composition. In anotherembodiment the iRNA agent is disposed within the device. In anotherembodiment, the device includes a mechanism to dispense a unit dose ofthe composition. In other embodiments the device releases thecomposition continuously, e.g., by diffusion. Exemplary devices includestents, catheters, pumps, artificial organs or organ components (e.g.,artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having thestructure or characteristics of a crystal, i.e., particles ofthree-dimensional structure in which the plane faces intersect atdefinite angles and in which there is a regular internal structure. Thecompositions of the invention may have different crystalline forms.Crystalline forms can be prepared by a variety of methods, including,for example, spray drying.

In one aspect the invention provides a method of modulating theexpression of a target gene in a cell, comprising providing to said cellan iRNA agent of this invention. In one embodiment, the target gene isselected from the group consisting of Factor VII, Eg5, PCSK9, TPX2,apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene,GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene,PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene,VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene,mutations in the p73 gene, mutations in the p21 (WAF1/CIP1) gene,mutations in the p27 (KIP1) gene, mutations in the PPM1D gene, mutationsin the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutationsin tumor suppressor genes, and mutations in the p53 tumor suppressorgene.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1. Synthesis of Carbohydrate Conjugate Building Blocks110 and 112

Preparation of 101:

Galactosamine pentaacetate 100 (52.00 g, 133.63 mmol) was taken indichloroethane (300 mL) at ambient temperature. TMSOTf (44.55 g, 200.44mmol) was added that and the mixture stirred at 50 C for 90 minutes in awater bath, heating stopped and the mixture stirred overnight at roomtemperature. It was poured in to an ice cold sodium bicarbonatesolution; extracted with dichloromethane, washed with water and driedover sodium sulfate. Solvents were removed the residue dried under highvacuum overnight to get the compound as dark gum (44.50 g,quantitative). It was used for next reaction with out any furtherpurification. ¹H NMR and MALDI confirmed the product formation. MS:Calculated for C₁₄H₁₉NO₈, 329.11. Found 352.1 (M+Na).

Preparation of 102:

Compound 101 (43.70 g, 133.56 mmol) and the benzyl ester (41.71 g,200.34 mmol) were dissolved in dichloroethane (300 mL), molecular sieves(50 g) was added to that and stirred for 30 minutes. TMSOTf (14.50 g,66.78 mmol) was added to that and the mixture stirred for overnight atroom temperature. It was poured in to an ice cold solution of sodiumbicarbonate and extracted with dichloromethane, washed with water anddried over sodium sulfate. Solvents were removed and the residuepurified by chromatography (gradient elution: 20-100%ethylacetate/hexanes) to get the required compound as light brown gummyliquid (60.50 g, 86%). 1HNMR, ¹³CNMR MS: Calculated for C₂₆H₃₅NO₁₁,537.22. Found 560.21 (M+Na).

Preparation 103:

Compound 102 (60.00 g, 111.68 mmol) was dissolved in a mixture ofMethanol/ethylacetate and degassed with argon. Pd/C (6.00 g, 10 wt %Degussa, wet type) was added and hydrogenated under balloon pressureovernight. Filtered through a small pad of celite; washed with methanoland dried under high vacuum overnight to get the product (48.85 g, 98%).1HNMR, ¹³CNMR MS: Calculated for C₁₉H₂₉NO₁₁, 447.17. Found 469.9 (M+Na).

Preparation of 104:

Compound 101 (42.30 g, 128.43 mmol) and the azido ethanol (26 g, 192.45mmol) were dissolved in dichloroethane (300 mL), molecular sieves (50 g)were added to that and stirred for 30 minutes. TMSOTf (14.29 g, 64.21mmol) was added to that and the mixture stirred for overnight at roomtemperature. It was poured in to an ice cold solution of sodiumbicarbonate and extracted with dichloromethane, washed with water anddried over sodium sulfate. Solvents were removed and the residuepurified by chromatography (gradient elution: 20-100% ethylacetate/hexanes, followed by 5-10% Methanol/ethylacetate) to get therequired compound as light brown gummy liquid (59.23 g, 91.00%). 1HNMR,¹³CNMR MS: Calculated for C₂₀H₃₂N₄O₁₁, 504.21. Found 527.1 (M+Na).

Preparation of 105:

Compound 104 (9.33 g, 18.50 mmol) was dissolved in THF (100 mL) to thatPPh₃ (5.97 g, 22.2 mmol) was added and the mixture stirred for 48 h. TLCchecked to see complete disappearance of starting material. Water (1 mL,55 mmol) and stirred for another 24 h. TFA (2.85 mL, 23.12 mmol) andtoluene (40 mL) were added and the solvents were removed under reducedpressure. The residue was co-evaporated with toluene (2×40 mL) two timesand dried under high vacuum. It was used for the next reaction in thesame day. MS: Calculated for C₂₀H₃₄N₂O₁₁, 478.22. Found 500.8 (M+Na).

Preparation of 107:

Compound 106 (JOC 2002) (6.94 g, 14.73 mmol) and monoboc propyl amine(10.26 g, 58.89 mmol) were dissolved in DMF (100 mL), to that HBTU(17.26 g, 45.50 mmol) and DIEA (15.36 mL, 88.14 mmol) were added andstirred overnight. Reaction mixture was poured in to ice-water mixtureand extracted with dichloromethane, washed with sodium bicarbonatesolution, brine and dried over sodium sulfate. Solvents were removed andthe residue was purified by chromatography (Ethyl acetate, followed by2-10% MeOH/DCM) to get the product as white fluffy solid (10.49 g, 76%).MS: Calculated for C₄₅H₇₇N₇O₁₄, 939.55. Found 940.53 (M+H).

Preparation of 108:

Compound 107 (2.40 g, 2.56 mmol) was dissolved in dichloromethane (10mL), to that a mixture of TFA/DCM (1:4, 10 mL) was added and stirred for30 minutes. Reaction was monitored by mass spectra. 100 mL of toluenewas added and removed the solvent under reduced pressure. The residuewas co-evaporated two times with toluene (2×100 mL) and dried under highvacuum to get the compound as its TFA salt (white gum, 2.47 g, 99%). Itwas used for the next reaction with out any further purification. MS:Calculated for C₃₀H₅₃N₇O₈, 639.40. Found 640.45 (M+H).

Preparation of 109:

GalNAc acid 103 (4.00 g, 8.99 mmol) was dissolved in DMF (50 mL); HBTU(3.75 g, 9.88 mmol), HOBt (1.34 g, 9.88 mmol) and DIEA (5 mL, 3.2 eq)was added to that and stirred for 3-4 minutes. A solution of 108 (2.47g, 2.50 mmol) in DMF was added to that and stirred the reaction mixtureovernight. TLC was checked, solvents were removed under reducedpressure. The residue was dissolved in dichloromethane, washed withsodium bicarbonate solution (50 mL), water (100 mL) and dried oversodium sulfate. Solvents were removed and the residue was purified bychromatography (ethyl acetate, followed by gradient elution 5-15%MeOH/DCM) to get the product 109 as a white solid (4.20 g, 87%). MS:Calculated for C₈₇H₁₃₄N₁₀O₃₈, 1926.89. Found 1949.5 (M+Na).

Preparation of 110:

GalNAc derivative 109 (7.50 g, 4.18 mmol) was taken in methanol (50 mL)degassed with argon. Pd/C (0.800 g, 10 wt % Degussa type wet) and coupleof drops of acetic acid were added; the mixture was hydrogenated underballoon pressure overnight. Reaction mixture was filtered through asmall pad of celite, washed with methanol. TFA (0.465 mL, 5.22 mmol) wasadded and removed the solvent under reduced pressure. The residue wasco-evaporated with toluene (2 times) and dried under high vacuumovernight to get the compound as TFA salt (pale yellow solid, 7.30 g,99%). MS: Calculated for C₇₉H₁₂₈N₁₀O₃₆, 1792.85. Found 1815.9 (M+Na).

Preparation of 111:

The tricarboxylic acid 106 (2.17 g, 4.625 mmol) and amine (18.50 mmol,crude from previous reaction) was dissolved in DMF (100 mL). To thatTBTU (5.34 g, 16.63 mmol), HOBt (2.24 g, 16.59 mmol) and DIEA (5.64 mL,32.36 mmol) was added and stirred the reaction mixture for 24 h. Afterstirring 24 hrs an additional amount of DIEA (4 mL) was added continuedstirring. After 48 hrs solvents were removed under reduced pressure, theresidue was dissolved in dichloromethane, washed with 1M phosphoric acidsolution, sodium bicarbonate solution, water and dried over sodiumsulfate. Solvents were removed and the residue was purified bychromatography (ethyl acetate, followed by 3-15% MeOH/DCM) to get therequired compound 111 as a white solid (5.80 g, 68%) MS: Calculated forC₈₁H₁₂₅N₇O₄₁, 1851.79. Found 1874.20 (M+Na).

Preparation of 112:

GalNAc derivative 111 (5.75 g, 3.09 mmol) was taken in methanol (100 mL)degassed with argon. Pd/C (0.600 g, 10 wt % Degussa type wet) and coupleof drops of acetic acid were added; the mixture was hydrogenated underballoon pressure for 36 hrs. Reaction mixture was filtered through asmall pad of celite, washed with methanol. TFA (0.354 mL, 1.25 eq) andtoluene (30 mL) was added and removed the solvent under reducedpressure. The residue was co-evaporated with toluene (2 times) and driedunder high vacuum overnight to get the compound as TFA salt (5.70 g,crude). MS: Calculated for C₈₁H₁₂₅N₇O₄₁, 1717.75. Found 1740.5 (M+Na).

Example 2. Synthesis of Carbohydrate Conjugate 118

Preparation of 115:

Hydroxy proline amine (3.00 g, 7.15 mmol) and Dodecanedioic acid monomethyl ester (1.748 g, 7.15 mmol) were taken together in DMF (50 mL). Tothat HBTU (3.25 g, 8.56 mmol) and DIEA (3.7 mL, 21.24 mmol) were addedand stirred the reaction over night. The reaction mixture was poured into ice water mixture and extracted with DCM. Washed with bicarbonatesolution, water, brine and dried over sodium sulfate. Solvent wasremoved and the residue was purified by chromatography (eluted with 50%ethyl acetate/hexane, ethyl acetate, followed by 5% MeOH/DCM) to get therequired compound 115 as white solid (4.30 g, 93%). MS: Calculated forC₃₉H₅₁NO₇, 645.37. Found 646.35 (M+H).

Preparation of 116:

Compound 115 (4.25 g, 6.58 mmol) was dissolved in a mixture ofTHF/DCM/Water (50 mL, 2:1:1). LiOH (1.90 g, 45.2 mmol) was added and themixture stirred overnight. TLC checked, acetic acid was added toneutralize the reaction mixture. Solvent was removed and the residueextracted with DCM. TEA (excess) added to the DCM solution and filteredthe solution through a small pad of silica gel to get the requiredproduct 116 as its TEA salt (4.15 g, 86%). MS: Calculated for C₃₈H₄₉NO₇,631.35. Found 630.34 (M−H).

Preparation of 117:

Compound 116 (1.30 g, 2.06 mmol) and HBTU (0.821 g, 1.05 eq.) were takentogether in DMF (30 mL). To that DIEA (1.07 ml, 3 eq) was added andstirred the reaction mixture for 3-4 minutes. A solution of amine 110(3.00 g, 1.58 mmol) was added followed by 1 eq. DIEA. The reactionmixture stirred overnight at room temperature. Solvents were removedunder reduced pressure. The residue dissolved in DCM, washed withbicarbonate and water. DCM layer was dried over sodium sulfate andremoved the solvents. The residue was purified by chromatography (elutedfirst with ethyl acetate, followed by 5-20% MeOH/DCM) to get the product117 as white solid (3.35 g, 88%). MS: Calculated for C₁₁₇H₁₇₅N₁₁O₄₂,2406.19. Found 2429.10 (M+Na).

Preparation of Solid Support 118:

Compound 117 (3.30 g, 1.37 mol), succinic anhydride (0.274 g, 2 eq) andDMAP (0.501 g, 3 eq.) were dissolved the DCM and stirred overnight.Reaction mixture was diluted with DCM, washed with water and cold dilutecitric acid solution. DCM layer was dried over sodium sulfate andremoved the solvent. The residue as filtered through a small pad ofsilica gel to the succinate as an off white solid (3.81 g) as its TEAsalt. MS: Calculated for C₁₂₁H₁₇₉N₁₁O₄₅, 2506.21. Found 2529.20 (M+Na).Succinate (2.20 g, 0.877 mmol) and HBTU (0.334 g, 0.877 mmol) weredissolved in DMF (100 mL). To that DIEA (0.457 mL, 2.62 mmol) was addedand swirl the reaction for 3-4 minutes. Polystyrene support (12.30 g)was added to that and shaken the mixture for 24 hrs. Filtered through afrit and washed with DCM, 10% MeOH/DCM, DCM and ether. Solid supportdried under vacuum for 2 hrs. It was capped with 25% Ac₂O/Py mixture for½ hr. The same washing and drying procedure repeated to the solidsupport 118 (13.10 g, 50.5 □mol/g loading).

Example 3. Synthesis of Carbohydrate Conjugate 122

Preparation of 119:

Z-amino caproic acid (2.19 g, 8.25 mmol) was dissolved in DMF (50 mL).To that HBTU (3.13 g, 8.25 mmol) and DIEA (7.19 mL, 5.00 eq.) was addedand stirred the mixture for few minutes. GalNAc amine 112 (10.10 g, 5.52mmol) was dissolved in 50 ml of DMF was added to that and stirred for 48hrs. TLC and MALDI were checked for product formation. Solvents wereremoved and the residue was dissolved in DCM, washed with NaHCO₃solution and water. Dried over sodium sulfate and removed the solventsunder reduced pressure. Residue was purified by chromatography (elutedwith ethyl acetate, followed by gradient elution of 5-15% MeOH/DCM) toget the required compound 119 as off white solid (6.20 g, 57%). MS:Calculated for C₈₇H₁₃₆N₈O₄₂, 1964.88. Found 1987.75 (M+Na).

Preparation of 120:

Compound 119 (6.10 g, 3.10 mmol) was dissolved in Methanol (50 mL), tothat 1 mL of acetic acid was added. Degassed the reaction mixture, Pd/C(0.700 g, 10 wt % Degussa wet type) was added to that and hydrogenatedunder balloon pressure for 36 hrs. Reaction mixture was filtered througha small pad of celite, washed with MeOH. To that 1.25 eq of TFA andtoluene (50 mL) were added and removed solvents under reduced pressure.The residue was co-evaporated with toluene two times and dried underhigh vacuum overnight night to get the required compound as an off whitesolid (6.10 g, quantitative). This compound used as such for the nextreaction with out any further purification. MS: Calculated forC₇₉H₁₃₀N₈O₄₀, 1830.84. Found 1853.81 (M+Na).

Preparation of 121:

Compound 116 (5.06 g, 6.90 mmol), GalNAc amine 112 (10.55 g, 5.756 mmol)TBTU (2.44 g, 1.1 eq.) and HOBt (1.025 g, 1.1 eq) were taken together inDMF (100 mL). To that DIEA (6 mL ml, 34.51 mmol) was added and stirredthe reaction mixture for 48 hrs. Reaction was monitored by TCL as wellas MALDI. Solvents were removed under reduced pressure. The residuedissolved in DCM, washed with bicarbonate and water. DCM layer was driedover sodium sulfate and removed the solvents. The residue was purifiedby chromatography (eluted first with ethyl acetate, followed by 3-10%MeOH/DCM) to get the product 121 as off white solid (10.50 g, 79%). MS:Calculated for C₁₁₁H₁₆₆N₈O₄₅, 2331.09. Found 2354.03 (M+Na).

Preparation of 122:

Compound 121 (2.00 g, 0.857 mmol), succinic anhydride (0.186 g, 2 eq),DMAP (0.314 g, 3 eq.) are taken together in DCM and stir overnight.Solvent is removed and the residue filter through a small pad of silicagel to get the succinate as its TEA salt. Succiniate (2.00 g, 0.857mmol) and HBTU (0.325 g, 0.857 mmol) are dissolved in DMF (100 mL). Tothat DIEA (0.450 mL, 2.57 mmol) is added and swirl the reaction for 3-4minutes. Polystyrene support (10.00 g) is added to that and shaken themixture for 24 hrs. Filter through a frit and washed with DCM, 10%MeOH/DCM, DCM and ether, it is capped with acetic anhydride to get thesolid support 122.

Example 4. Synthesis of Carbohydrate Conjugate 128

Preparation of 125:

Amine 123 (2.75 g, 4.61 mmol) and Mono ethyl hexane dioic acid (0.886 g,5.09 mmol) were dissolved in DMF (50 mL). To that HBTU (2.09 g, 5.51mmol) and DIEA (2.88 mL, 16.53 mmol) were added and stirred the reactionmixture overnight. Reaction mixture was poured in to an ice watermixture and extracted with DCM, washed with bicarbonate solution anddried over sodium sulfate. Solvent was removed and the residue waspurified by chromatography (eluted with 50% EtOAc/Hexane, EtOAc,followed by 5-10% MeOH/DCM) to get the required product as a fluffywhite solid (2.25 g, 65%). MS: Calculated for C₄₀H₅₂N₂O₈S2, 752.32.Found 753.31 (M+Na).

Preparation of 126:

Compound 125 (2.20 g, 2.97 mmol) was dissolved in a mixture of THF/Water(20 mL, 2:1). LiOH (0.187 g, 4.45 mmol) was added and the mixturestirred 4 hrs. Reaction was monitored TLC, after 4 hrs, cooled andcitric acid was added to quench the reaction mixture. Solvent wasremoved and the residue was extracted DCM, washed with water. Dried oversodium sulfate and removed the solvent. The residue was purified bychromatography (EtOAc, 3-20% MeOH/DCM) to get the required product 126(0.750 g, 35%) as its TEA salt. MS: Calculated for C₃₈H₄₈N₂O₈S2, 724.29.Found 723.28 (M−H).

Preparation of 127:

Compound 126 (1.008 g, 1.390 mmol), 110 (1.904 g, 1.007 mmol) and HBTU(0.400 g, 1.054 mmol) were dissolved in DMF (20 mL). To that DIEA (0.525mL, 3 eq.) was added and stirred the reaction for 2 days. Reactionmixture was monitored by TLC and MALDI. Solvents were removed and theresidue dissolved in DCM, washed with water and bicarbonate solution.DCM layer was dried over sodium sulfate and removed the solvent. It wasthen purified by chromatography (first ethyl acetate, followed by 3-15%MeOH/DCM) to get the required product as a fluffy off white solid (1.90g, 76%). MS: Calculated for C₁₁₇H₁₇₄N₁₂O₄₃S₂, 2499.12. Found 2522.12(M+Na).

Preparation of Solid Support 128:

Compound 127 (2.00 g, 0.800 mmol), succinic anhydride (0.160 g, 2 eq),DMAP (0.300 g, 3 eq.) are taken together in DCM and stir overnight.Solvent is removed and the residue filter through a small pad of silicagel to get the succinate as its TEA salt. Compound 127 (2.00 g, 0.769mmol) and HBTU (0.290 g, 0.769 mmol) are dissolved in DMF (100 mL). Tothat DIEA (0.500 mL, 3 mmol) is added and swirl the reaction for 3-4minutes. Polystyrene support (10.00 g) is added to that and shaken themixture for 24 hrs. Filter through a frit and washed with DCM, 10%MeOH/DCM, DCM and ether, it is capped with acetic anhydride to get thesolid support 128.

Example 5. Synthesis of Carbohydrate Conjugate 136

Preparation of 131:

Mannose trichloroacetimidate 129 (15.00 g, 20.24 mmol) and azido alcohol(4.25 g, 1.2 eq) were dissolved in Toluene and aziotroped two times. Theresidue dried under high vacuum overnight. Anhy. diethyl ether (30 mL)and Molecular sieves (10 g) were added to that. Reaction mixture cooledin an ice-water bath. TMSOTf (0.5 mL, 0.1 eq) was added to that andstirred the mixture for 10 minutes. Reaction was monitored by TLC andquenched with TEA. Filtered of the molecular sieves and solvents wereremoved under reduced pressure. Residue was purified by chromatography(20-50% EtOAc/Hexane) to get compound as colorless liquid (8.36 g, 55%).MS: Calculated for C₄₀H₃₉N₃O₁₂, 753.25. Found 776.23 ((M+Na).

Preparation of 132:

Compound 131 (8.30 g, 11.01 mmol) was dissolved in anhy. THF (70 mL), tothat PPh3 (3.46 g, 1.2 eq) was added and the mixture stirred for twodays at ambient temperature. Water (1 mL) was added to that and stirredthe mixture for another 24 hrs. Reaction was monitored by TLC. Trifluroacetic acid (1.06 mL, 1.25 eq) and toluene (50 mL) was added to that.Solvents were removed under reduced pressure and residue wasco-evaporated toluene two times and dried under high vacuum. This usedas such for the next reaction without further purification. MS:Calculated for C₄₀H₄₁NO₁₂, 727.26. Found 750.23 ((M+Na).

Preparation of 133:

Tricarboxylic acid (11.05 g, 23.45 mmol), and amine (68.19 g, 94 mmol,crude from previous reaction) was dissolved in DMF (200 mL). To thatTBTU (27.09 g, 84 mmol), HOBt (11.34 g, 84 mmol) and DIEA (28 mL, 160mmol) was added and stirred the reaction mixture for 24 h. Afterstirring 24 hrs an additional amount of DIEA (28 mL) was added continuedstirring. After 48 hrs solvents were removed under reduced pressure, theresidue was dissolved in dichloromethane, washed with 1M phosphoric acidsolution, sodium bicarbonate solution, and water and dried over sodiumsulfate. Solvents were removed and the residue was purified bychromatography (ethyl acetate, followed by 3-15% MeOH/DCM) to get therequired compound 133 as a white solid (41.95 g, 67%) MS: Calculated forC₁₄₁H₁₄₆N₄O₄₄, 2598.93. Found 2621.89 (M+Na).

Preparation of 134:

Compound 133 (3.05 g, 1.176 mmol) was dissolved in a mixture ofDCM/MeOH. To that 50 eq. of ammoniumformate was added followed by 5%Pd/C (1.5 g, 50 wt %) and stirred for 8 hrs at ambient temperature. Itwas filtered through small pad of celite, washed with MeOH/DCM, solventwas removed and residue dried under high vacuum over night to thecompound as a white solid (2.65 g, 92%). MS: Calculated forC₁₃₃H₁₄₀N₄O₄₂, 2464.89. Found 2487.92 (M+Na).

Preparation of 135:

Mannose amine (2.076 g, 0.842 mmol), 116 (0.740 g, 1.00 mmol) and TBTU(0.0.353 g, 1.1 eq.) and HOBt (0.149 g, 1.1 eq) were dissolved in DMF(30 mL). To that DIEA (0.0.869 mL, 5 eq.) was added and stirred thereaction for 2 days. Reaction mixture was monitored by TLC and MALDI.Solvents were removed and the residue dissolved in DCM, washed withwater and bicarbonate solution. DCM layer was dried over sodium sulfateand removed the solvent. It was then purified by chromatography (firstethyl acetate, followed by 2-4% MeOH/DCM) to get the required productsas a fluffy off white solid (1.48 g, 57%). MS: Calculated forC₇₁H₁₈₇N₅O₄₈, 3078.23. Found 3101.25 (M+Na).

Preparation of Solid Support 136:

Compound 117 (2.10 g, 0.681 mmol), succinic anhydride (0.136 g, 2 eq)and DMAP (0.249 g, 3 eq.) were dissolved the DCM and stirred overnight.Reaction mixture was diluted with DCM, washed with water and cold dilutecitric acid solution. DCM layer was dried over sodium sulfate andremoved the solvent. The residue as filtered through a small pad ofsilica gel to the succinate as an off white solid (1.56 g) as its TEAsalt. MS: Calculated for C₁₇₅H₁₉₁N₅O₅₁, 3178.25. Found 3201.20 (M+Na).Succinate (1.00 g, 0.305 mmol) and HBTU (0.138 g, 1.2 eq.) weredissolved in DMF (100 mL). To that DIEA (0.50 mL, excess) was added andswirl the reaction for 3-4 minutes. Polystyrene support (6.05 g) wasadded to that and shaken the mixture for 24 hrs. Filtered through a fritand washed with DCM, 10% MeOH/DCM, DCM and ether. Solid support driedunder vacuum for 2 hrs. It was capped with 25% Ac₂O/Py mixture for ½ hr.The same washing and drying procedure repeated to the solid support 136(6.70 g, 42 μmol/g loading).

Example 6. Synthesis of Carbohydrate Conjugate 143

Preparation of 138:

Mannose trichloroacetimidate 129 (15.23 g, 20.55 mmol) and 137 (4.36 g,1.02 eq.) were dissolved in Toluene and aziotroped two times. Theresidue dried under high vacuum overnight. Anhy. diethyl ether (30 mL)and Molecular sieves (10 g) were added to that. Reaction mixture cooledin an ice-water bath. TMSOTf (0.5 mL, 0.1 eq) was added to that andstirred the mixture for 10 minutes. Reaction was monitored by TLC andquenched with TEA. Filtered of the molecular sieves and solvents wereremoved under reduced pressure. Residue was purified by chromatography(hexane, 15-25% EtOAc/Hexane) to get compound as colorless liquid (14.52g, 90%). MS: Calculated for C₄₆H₄₂O₁₂, 786.27. Found 809.25 ((M+Na).

Preparation of 139:

Mannose benzyl ester (14.30 g, 18.17 mmol) was dissolved in Ethylacetate (100 mL) to that two drops of acetic acid was added. Degassed,Pd/C (1.50 g, 10 wt % Degussa wet type) was added and hydrogenated underballoon pressure for 24 hrs. Reaction was monitored by TLC and MALDI. Itwas filtered through a small pad of celite, washed with ethyl acetate.Solvent was removed and the residue dried under high vacuum to get thecompound as color less oil (11.20 g, 90%). MS: Calculated for C₃₉H₃₆O₁₂,696.22. Found 719.18 ((M+Na).

Preparation of 141:

Hydroxy Proline amine 140 (3.82 g, 7.18 mmol), 141 (5.00 g, 7.18 mmol)and HBTU (2.65 g, 7.18 mmol) were dissolved in DMF (50 mL). To that DIEA(3.65 mL, 5 eq.) was added and stirred the reaction for 3 hrs. Reactionmixture was monitored by TLC. Solvents were removed and the residuedissolved in DCM, washed with water and bicarbonate solution. DCM layerwas dried over sodium sulfate and removed the solvent. It was thenpurified by chromatography (first ethyl acetate, followed by 5-10%MeOH/EtOAc) to get the required product as a white solid (4.08 g, 46%).MS: Calculated for C₇₁H₇₄N₂O₁₆, 1210.50. Found 1233.40 (M+Na).

Preparation of Solid Support 143:

Compound 141 (2.00 g, 1.652 mmol), succinic anhydride (0.330 g, 2 eq),DMAP (0.604 g, 3 eq.) are taken together in DCM and stir overnight.Solvent is removed and the residues filter through a small pad of silicagel to get the succinate as its TEA salt 142. Succiniate (2.00 g, 1.526mmol) and HBTU (0.578 g, 1.526 mmol) are dissolved in DMF (100 mL). Tothat DIEA (1.32 mL, 5 eq.) is added and swirl the reaction for 3-4minutes. Polystyrene support (10.00 g) is added to that and shaken themixture for 24 hrs. Filter through a frit and washed with DCM, 10%MeOH/DCM, DCM and ether, it is capped with acetic anhydride to get thesolid support 143.

Example 7. Synthesis of Carbohydrate Conjugate 152

Preparation of 146:

Compound 144 (26.55 g, 64.06 mmol) and 145 (10.00 g, 53.43 mmol) weredissolved in DMF (150 mL). To that HBTU (24.12 g, 64 mmol) and DIEA (46mL, 5 eq) were added and stirred the reaction mixture overnight. TLCchecked and the mixture was added to ice cold water and extracted with amixture of ether and ethyl acetate dried over sodium sulfate. Solventswere removed and the crude product was purified by chromatography(20-50% ethylacetate/Hexane) to get the required product as an off whitesolid (23.20 g, 74%). MS. MW calc. for C₃₂H₄₅N₃O₇: 583.72. Found 584.73(M+H).

Preparation of 147:

Compound 146 (3.30 g, 5.65 mmol) was dissolved in a mixture of ethylacetate/MeOH and hydrogenated under balloon pressure using Pd/C (500 mg)as catalyst overnight. Filtered through a small pad of celite andremoved the solvent, this product used for the next reaction withoutfurther purification. MS. MW calc. for C₁₆H₃₃N₃O₃: 315.25. Found 316.26(M+H).

Preparation of 148:

Compound 147 (5.65 mmol) and GalNAc acid 103 (5.81 g, 12.99 mmol) weredissolved in DMF (80 mL). To that HBTU (4.97 g, 13.10 mmol) and DIEA(7.00 mL, 3 eq) were added and stirred the reaction mixture overnight.Solvents were removed and the residue dissolved in DCM and washed withwater and brine, dried over sodium sulfate. Solvents were removed andthe crude product was purified by chromatography (EtOAc, followed by3-10% MeOH/DCM) to get the required product as an off white solid (5.25g, 79%). MS. MW calc. for C₅₄H₈₇N₅O₂₃: 1173.58. Found 1196.60 (M+Na).

Preparation of 149:

Biantineary GalNAc derivative 148 (5.15 g, 4.40 mmol) was dissolved in15 mL of anhydrous DCM, to that 3 mL of anisole and 30 mL of TFA wereadded and stirred the reaction mixture for 2 hrs at ambient temperature.TLC checked and toluene was added to the reaction mixture, removed thesolvents under reduced pressure. Co-evaporated with toluene two timesand the residue dissolved in DCM, washed with water, dried overanhydrous sodium sulfate. Crude product was purified by filtrationcolumn (10% MeOH/DCM) to get the required product as pale brown solid(4.40 g, 91%). MS. MW calc. for C₅₀H₇₉N₅O₂₃: 1117.52. Found 1140.62(M+Na).

Preparation of 150:

Biantineary GalNAc acid 149 (4.30 g, 3.84 mmol) and hydroxyl prolineamine 153 (2.25 g, 1.1 eq) were dissolved in DMF (50 mL). To that HBTU(1.46 g, 3.84 mmol) and DIEA (3.3 mL) were added and stirred thereaction mixture for 3 hrs. Solvents were removed and the residuedissolved in DCM, washed with water and bicarbonate, dried over sodiumsulfate. Solvents were removed and the crude product purified bychromatography (3-10% MeOH/DCM) to get the required product as whitesolid (3.25 g, 52%). MS. MW calc. for C₈₂H₁₁₇N₇O₂₇: 1631.80. Found1654.45 (M+Na).

Preparation of 151:

Compound 150 (3.30 g, 2.02 mmol), succinic anhydride (0.404 g, 2 eq),DMAP (0.740 g, 3 eq.) are taken together in DCM (30 mL) and stirovernight. Solvent is removed and the residues filter through a smallpad of silica gel to get the succinate as its TEA salt 151. MS. MW calc.for C₈₆H₁₂₁N₇O₃₀: 1731.82. Found 1753.87 (M+Na).

Preparation of Solid Support 152:

Succinate 151 (2.02 mmol) and HBTU (0.842 g, 1.1 eq.) were dissolved inDMF (100 mL). To that DIEA (1.50 mL, excess) was added and swirl thereaction for 3-4 minutes. Polystyrene support (28 g) was added to thatand shaken the mixture overnight. Filtered through a frit and washedwith DCM, 10% MeOH/DCM, DCM and ether. Solid support dried under vacuumfor 2 hrs. It was capped with 25% Ac₂O/Py mixture for ½ hr. The samewashing and drying procedure repeated to the solid support 152 (30.10 g,30 μmol/g loading).

Example 8. Synthesis of Carbohydrate Conjugate 161

Preparation of 155:

Hydroxy proline amine 153 (10.00 g, 18.76 mmol) and 154 (4.98 g, 18.76mmol) were dissolved in DMF (100 mL). To that HBTU (7.83 g, 20.64 mmol)and DIEA (9.81 mL, 56.29 mmol) were added and stirred the reaction for 2hrs. TLC checked and the mixture was added to ice cold water andextracted with a mixture of ether and ethyl acetate dried over sodiumsulfate. Solvents were removed and the crude product was purified bychromatography (0-15% MeOH/DCM) to get the required product as an offwhite solid (13.20 g, 90%). MS. MW calc. for C₄₆H₅₇N₃O₈: 779.41. Found780.42 (M+H).

Preparation of 156:

Compound 155 (13.00 g, 16.66 mmol) was dissolved in a mixture of ethylacetate/MeOH and hydrogenated under balloon pressure using Pd/C (1.50 g)as catalyst overnight in presence of small amount of triethyl amine.Filtered through a small pad of celite and removed the solvent, thisproduct used for the next reaction without further purification (9.93 g,92%). MS. MW calc. for C₃₈H₅₁N₃O₆: 645.38. Found 646.40 (M+H).

Preparation of 157:

Compound 156 (9.90 g, 15.33 mmol) and diCbz lysine (6.36 g, 15.33 mmol)were dissolved in DMF (100 mL). To that HBTU (6.11 g, 15.33 mmol) andDIEA (8 mL, excess) were added and stirred the reaction for 2 hrs. TLCchecked and the mixture was added to ice cold water and extracted with amixture of ether and ethyl acetate dried over sodium sulfate. Solventswere removed and the crude product was purified by chromatography (0-10%MeOH/DCM) to get the required product as an off white solid (13.10 g,83%). MS. MW calc. for C₆₀H₇₅N₅O₁₁: 1041.55. Found 1042.57 (M+H).

Preparation of 158:

Compound 157 (12.90 g, 12.37 mmol) was dissolved in a mixture of ethylacetate/MeOH and hydrogenated under balloon pressure using Pd/C (1.30 g)as catalyst. TLC checked after 3 hrs filtered through a small pad ofcelite and removed the solvent, this product used for the next reactionwithout further purification. MS. MW calc. for C₄₄H₆₃N₅O₇: 773.47. Found774.50 (M+H).

Preparation of 160:

Compound 158 (2.32 g, 3 mmol) and Glucose acid 159 (4.50 g 6.45 mmol)were dissolved in DMF (60 mL). To that HBTU (2.44 g, 6.45 mmol) and DIEA(3.36 mL, 3 eq) were added and stirred the reaction for 2 hrs and pouredthe reaction mixture to ice cold water and extracted with EtOAc/DCM,dried over sodium sulfate. Solvents were removed and the crude productwas purified by chromatography (EtOAc, followed by 0-10% MeOH/DCM) toget the required product as an off white solid (5.40 g, 85%). MS. MWcalc. for C₁₂₂H₁₃₁N₅O₂₉: 2129.89. Found 2152.90 (M+Na).

Preparation of Solid Support 161:

Compound 160 (5.20 g, 2.44 mmol), succinic anhydride (0.488 g, 2 eq) andDMAP (0.894 g, 3 eq.) were dissolved the DCM and stirred overnight.Reaction mixture was diluted with DCM, washed with water and cold dilutecitric acid solution. DCM layer was dried over sodium sulfate andremoved the solvent. The residue as filtered through a small pad ofsilica gel to the succinate as an off white solid as its TEA salt. MS:MW calc. for C₁₂₆H₁₃₅N₅O₃₂: 2229.91. Found 2252.50 (M+Na). Succinate(2.44 mmol) and HBTU (0.925 g, 1.2 eq.) were dissolved in DMF (200 mL).To that DIEA (1.27 mL, excess) was added and swirl the reaction for 3-4minutes. Polystyrene support (24 g) was added to that and shaken themixture for 24 hrs. Filtered through a frit and washed with DCM, 10%MeOH/DCM, DCM and ether. Solid support dried under vacuum for 2 hrs. Itwas capped with 25% Ac₂O/Py mixture for ½ hr. The same washing anddrying procedure repeated to the solid support 161 (27 g, 31 umol/gloading).

Example 9. Synthesis of Carbohydrate Conjugate 165 and 166

Preparation of 163:

Compound 158 (5.40 g, 6.97 mmol) and mannose acid 139 (9.96 g 14.30mmol) were dissolved in DMF (100 mL). To that HBTU (5.42 g, 14.30 mmol)and DIEA (7.45 mL, excess) were added and stirred the reaction for 2 hrsand poured the reaction mixture to ice cold water and extracted withEtOAc/DCM, dried over sodium sulfate. Solvents were removed and thecrude product was purified by chromatography (EtOAc, followed by 2-10%MeOH/DCM) to get the required product as an off white solid (9.20 g,62%). MS. MW calc. for C₁₂₂H₁₃₁N₅O₂₉: 2129.89. Found 2152.65 (M+Na).

Preparation of Solid Support 165:

Compound 163 (3.20 g, 1.408 mmol), succinic anhydride (0.2835 g, 2 eq)and DMAP (0.516 g, 3 eq.) were dissolved the DCM and stirred overnight.Reaction mixture was diluted with DCM, washed with water and cold dilutecitric acid solution. DCM layer was dried over sodium sulfate andremoved the solvent. The residue as filtered through a small pad ofsilica gel to the succinate as an off white solid as its TEA salt. MS:MW calc. for C₁₂₆H₁₃₅N₅O₃₂: 2229.91. Found 2252.90 (M+Na). Succinate(1.408 mmol) and HBTU (0.640 g, 1.2 eq.) were dissolved in DMF (200 mL).To that DIEA (1.22 mL, excess) was added and swirl the reaction for 3-4minutes. Polystyrene support (20 g) was added to that and shaken themixture for 24 hrs. Filtered through a frit and washed with DCM, 10%MeOH/DCM, DCM and ether. Solid support dried under vacuum for 2 hrs. Itwas capped with 25% Ac₂O/Py mixture for ½ hr. The same washing anddrying procedure repeated to the solid support 161 (23.2 g, 54.7 umol/gloading).

Preparation of 166:

Compound 163 (4.01 g, 1.88 mmol) was dissolved in DCM (50 mL) and DIEA(0.65 mL, 3.75 mmol) was added. Amidite reagent (0.629 mL, 2.822 mmol)was added to this mixture and stirred the reaction mixture for 15minutes. TLC checked and transferred the reaction mixture to aseparatory funnel, washed with water and sodium bicarbonate solution.Dried over anhydrous sodium sulfate and removed the solvent. The crudeproduct was purified by chromatography (30-80% Acetone/DCM) to get theproduct (4.20 g, 96%). ³¹P NMR (CDCl₃, 400 MHz) δ=148.19, 147.79,147.33. MS. MW calc. for C₁₃₁H₁₄₈N₇O₃₀P: 2330.00. Found 2353.20 (M+Na).

Example 10. Synthesis of Carbohydrate Conjugate Building Blocks

Synthesis of 171, 172, 173 and 174.

Building blocks 171 and 172 are synthesized using a procedure similar tothat for synthesis of 103. Building blocks 173 and 174 are synthesizedusing a procedure similar to that for synthesis of 105.

Synthesis of 180.

Building block 180 is synthesized using a procedure similar to that forsynthesis of 110.

Synthesis of Building Block 188.

Example 11. Synthesis of Carbohydrate Conjugates

The building block 180 is coupled with amines 183, 185 and 188 toprovide carbohydrate conjugates 189, 190 and 191 respectively.

Preparation of 201:

Mannose (10.00 g, 55.53 mmol) and Decinol (100 g, solvent) and CSA (500mg) were stirred at 110° C. in an oil bath for overnight. The color ofthe decinol turned to dark brown overnight. Bulk of the decinol wasdistilled out under reduced pressure. The residue was dissolved in DCMand neutralized with TEA. Extracted the solution with water and driedover sodium sulfate. Solvent was removed and the residue was purified byfiltration through a small pad of silica gel, first ethyl acetatefollowed by 10-15% MeOH/DCM to get the product (7.52 g, 42%). ¹H NMR(CDCl₃, 400 MHz) δ=5.90-5.75 (m, 1H), 5.02-4.85 (m, 2H), 4.00-3.30 (m,7H), 2.10-1.94 (m, 2H), 1.60-1.49 (m, 2H), 1.41-1.20 (m, 12H).

Preparation of 203:

Compound 201 (0.172 g, 0.541 mmol) was dissolved in anhydrous DCM (10mL) under argon. MS was added to that and cooled the reaction in an icebath. BF₃.Et₂O (10 μl) was added to the reaction mixture with stirring.Galactose trichloroacetimidate 202 (1.00 g. 1.35 mmol) in 5 mL of DCMwas added drop wise over a period of 15 minutes. Reaction was monitoredby TLC, once the acceptor was finished the reaction was quenched withTEA and diluted with DCM, filtered off MS and dried. The residue waspurified by chromatography (gradient elution 10-40% EtOAc/Hexane) to thecompound as a white fluffy solid (0.550 g, 69%). ¹H NMR (CDCl₃, 400 MHz)δ=7.95-7.20 (m, 40H), 5.90-5.50 (m, 7H), 5.35 (d, J=8.05 Hz, 1H), 5.17(d, J=8.06 Hz, 1H), 4.98-4.81 (m, 3H), 4.65-4.09 (m, 9H), 3.81-3.42 (m,5H), 3.20 (bs, 1H), 2.79 (bs, 1H), 2.01-1.88 (m, 2H), 1.30-0.92 (m,12H). ¹³C NMR (CDCl₃, 100 MHz) δ=166.28, 166.20, 165.88, 165.76, 165.66,165.64, 165.40, 139.34, 134.04, 133.82, 133.71, 133.66, 133.42, 133.30,130.21, 129.99, 129.86, 129.70, 129.59, 129.28, 129.03, 129.00, 128.94,128.77, 128.73, 128.63, 128.61, 128.54, 128.47, 128.44, 114.37, 102.74,102.68, 98.81, 85.27, 72.43, 71.96, 71.37, 71.31, 71.01, 70.30, 70.26,70.05, 68.31, 68.23, 67.41, 66.11, 62.63, 62.08, 33.96, 29.65, 29.58,29.53, 29.58, 29.08, 26.20. MS. Molecular weight calculated forC₈₄H₈₂O₂₄, Cal. 1474.52. Found 1497.60 (M+Na).

Preparation of 204:

Compound 203 (0.104 g, 0.07 mmol) was dissolved in a mixture of DCM/Py(10 mL, 1:1). Ac₂O (0.5 mL, excess) and DMAP (0.050 g) and stirred thereaction overnight. The reaction was quenched with MeOH, solvents wereremoved and residue was purified by chromatography (gradient elution10-30% EtOAc/Hexane) to the compound was white fluffy solid (0.108 g,99%). ¹H NMR (CDCl₃, 400 MHz) δ=8.10-7.20 (m, 40H), 5.99 (dd, J=3.1, 7.8Hz, 2H), 5.88-5.75 (m, 2H), 5.70 (dd, J=7.82, 10.43 Hz, 1H), 5.65-5.47(m, 2H), 5.10-4.07 (m, 13H), 3.90-3.80 (m, 1H), 3.69-3.61 (m, 1H),3.36-3.28 (m, 1H), 2.98-2.81 (m, 1H), 2.08 (s, 3H), 2.10-2.01 (m, 4H),1.35 (s, 3H), 1.42-1.20 (m, 12H). ¹³C NMR (CDCl₃, 100 MHz) δ=170.12,170.08, 166.16, 165.67, 165.64, 165.48, 165.46, 164.78, 139.29, 133.80,133.70, 133.70, 133.54, 133.44, 133.41, 133.35, 130.13, 130.02, 129.92,129.69, 129.58, 129.49, 129.40, 129.15, 129.10, 128.88, 128.83, 128.79,128.73, 128.66, 128.47, 128.40, 114.35, 102.32, 99.58, 96.64, 74.51,72.11, 71.91, 71.46, 71.21, 69.78, 69.72, 69.51, 69.28, 68.19, 68.03,67.82, 67.12, 61.97, 61.83, 33.94, 29.63, 29.61, 29.55, 29.49, 29.27,29.20, 29.05, 26.11, 21.06, 20.02. MS: Molecular weight calculated forC₈₈H₈₆O₂₆, Cal. 1558.54. Found 1581.8 (M+Na).

Preparation of 205:

Compound 205 (1.36 g, 0.873 mmol) was dissolved in a mixture of Dioxane:Water (40 mL, 3:1). To the reaction mixture lutidine (0.203 mL, 2 eq),followed by OsO₄ solution (1 mL. 0.05M solution in ^(t)Butanol) wereadded. Sodium periodate (0.774 g, 4 eq) was added and stirred for 4 hr'sat room temperature. Reaction was monitored by TLC, once the startingmaterial was consumed; the mixture was diluted with water and extractedwith DCM (3 times) and dried over sodium sulfate. All the solvents wereremoved and the residue was directly used next reaction. Residue fromthe above reaction was dissolved in DMF (20 mL) to that Oxone (0.590 g,1.05 eq) and stirred at ambient temperature for 3 h. Once the startingmaterial was consumed, 2 mL of 1M HCl was added and diluted with Ethylacetate. Washed with water, brine and dried over sodium sulfate.Solvents were removed and the residue was purified by chromatography(gradient elution 20-40% EtOAc/hexane) to get the compound as a whitesolid (1.08 g 79%). ¹H NMR (DMSO-d₆, 400 MHz) δ=11.96 (s, 1H), 8.00-7.23(m, 40H), 5.85 (d, J=3.41 Hz, 1H), 5.82 (d, J=3.17 Hz, 1H), 5.79-5.63(m, 2H), 5.56 (dd, J=8.00, 10.01 Hz, 1H), 5.41 (dd, J=8.00, 10.01 Hz,1H), 5.25 (d, J=7.8 Hz, 1H), 5.15 (d, J=7.8 Hz, 1H), 4.90-4.35 (m, 7H),4.10-3.55 (m, 4H), 3.30-3.20 (m, 1H), 2.96-2.87 (m, 1H), 2.18-2.10 (m,2H), 1.96 (s, 3H), 2.01-1.95 (m, 1H), 1.51-1.39 (m, 2H), 1.27 (s, 3H),1.20-1.01 (m, 12H). ¹³C NMR (CDCl₃, 100 MHz) δ=178.68, 178.48, 170.26,170.16, 166.25, 165.78, 165.73, 165.70, 165.54, 165.53, 164.83, 133.85,133.75, 133.60, 133.49, 130.18, 130.08, 128.85, 129.61, 129.52, 129.44,129.20, 129.13, 128.91, 128.89, 128.81. 128.78, 128.71, 128.51, 128.45,102.34, 99.67, 96.65, 74.60, 72.17, 71.94, 71.49, 71.21, 69.82, 69.79,69.59, 69.37, 68.22, 68.11, 67.81, 67.20, 64.55, 61.99, 61.85, 60.59,44.06, 33.96, 30.79, 29.39, 29.31, 29.24, 29.20, 29.17, 29.08, 26.08,24.85, 24.79, 22.20, 21.24, 21.11, 20.07.

MS: Molecular weight calculated for C₈₇H₈₄O₂₈, Cal. 1576.51. Found1599.50 (M+Na).

Preparation of 206:

Compound 205 (0.850 g, 0.539 mmol), hydroxyl proline amine (0.300 g,0.563 mmol) and HBTU (0.265 g, 0.698 mmol) were dissolved in DMF underargon. DIEA (0.281 mL, 3 eq.) was added to that and stirred for 3 hrs atambient temperature. The reaction was monitored by TLC; once thestarting material was consumed the mixture was poured in to an ice watermixture; extracted with ethyl acetate washed with water, brine and driedover sodium sulfate. Solvents was removed and the residue was purifiedby chromatography (first ethyl acetate followed by a gradient elution3-10% MeOH/DCM) to get the product as a pale yellow solid (1.09 g, 96%).¹H NMR (CDCl₃, 400 MHz) δ=8.00-7.10 (m, 53H), 6.90-6.80 (m, 4H), 5.85(d, J=3.41 Hz, 1H), 5.82 (d, J=3.17 Hz, 1H), 5.79-5.63 (m, 2H), 5.56(dd, J=8.00, 10.01 Hz, 1H), 5.41 (dd, J=8.00, 10.01 Hz, 1H), 5.25 (d,J=7.8 Hz, 1H), 5.15 (d, J=7.8 Hz, 1H), 4.97 (d, J=4.15 Hz, 1H),4.90-4.80 (m, 3H), 4.70-4.30 (m, 7H), 4.20-4.00 (m, 2H), 3.95-3.85 (m,2H), 3.70 (s, 6H), 3.69-3.50 (m, 1H), 3.30-3.20 (m, 2H), 2.96-2.87 (m,1H), 2.18-2.10 (m, 2H), 1.96 (s, 3H), 2.01-1.95 (m, 1H), 1.51-1.39 (m,2H), 1.27 (s, 3H), 1.20-1.01 (m, 20H). ¹³C NMR (CDCl₃, 100 MHz)δ=171.87, 170.85, 169.46, 169.04, 165.25, 165.21, 165.09, 164.95,164.48, 164.53, 162.29, 158.09, 157.97, 145.08, 135.87, 135.73, 134.04,133.74, 133.56, 129.60, 129.18, 129.06, 128.91, 128.84, 128.81, 128.75,128.67, 128.63, 128.52, 128.41, 127.77, 127.58, 113.19, 113.09, 102.30,99.60, 96.60, 85.10, 75.68, 71.48, 70.02, 69.81, 68.99, 68.58, 66.55,61.86, 6=54.96, 45.74, 38.27, 36.32, 35.76, 35.46, 34.15, 30.74, 28.69,26.20, 25.34, 26.20, 25.34, 24.15, 20.48, 19.54. MS: Molecular weightcalculated for C₁₁₉H₁₂₂N₂O₃₂, Cal. 2090.80. Found 2013.90 (M+Na).

Preparation of Long Alkyl Chain CPG 207:

Hydroxy derivative 206 (0.550 g, 0.263 mmol) was dissolved in DCM (10mL) to that Succinic anhydride (0.078 g, 3 eq) and DMAP (0.128 g, 4 eq.)were added and stirred overnight. TLC showed completion of reaction. Thereaction mixture was diluted with DCM (20 mL), washed successively withcold dilute citric acid and water (2 times), dried over sodium sulfate.Solvents were removed and dried under high vacuum to get the succinate.PPh₃ (0.90 g, 1.3 eq.), DMAP (0.048 g, 1.5 eq.) and the succinate fromthe previous step were dissolved in a mixture of acetonitrile and DCM (6mL). A solution of DTNP (0.086 g, 1.05 eq.) in DCM (1 mL) was added tothe above solution. The mixture was slowly shaken for 3-4 minutes. Longchain alkyl amine-CPG (lcaa CPG, 1.40 g, 133 μmol/g) was added to themixture and gently shaken for 2 h. The CPG was filtered, successivelywashed with DCM, mixture of MeOH/DCM (1:9) and DCM until filtrateremained colorless and dried. The dried CPG was transferred into anotherflask treated with Ac₂O in pyridine (25%) in the presence of TEA (1 mL)for 15 min. under gentle shaking. Finally the CPG was filtered, washedwith DCM, DCM:MeOH (9:1), followed by DCM and ether. The CPG 207 wasdried under vacuum overnight and the loading was measured as reported(1.48 g, loading 36 μmol/g).

Compound 217 was synthesized according to the reported procedure(Martin, C.; Karen, P.; Laurence, V. Chem. Pharm. Bull. 2004, 52,965-971.)

Preparation of 218:

1-Decinol (0.300 g, 1.92 mmol) and trichloroacetimidate 217 (2.33 g, 1.2eq) was dissolved in anhydrous DCM (10 mL) under argon. MS was added tothat and cooled the reaction in an ice bath. BF₃.Et₂O (30 μl) was addedto the reaction mixture with stirring. Reaction was monitored by TLC,once the donor reacted the reaction was quenched with TEA and dilutedwith DCM, filtered off MS and dried. The residue was purified bychromatography (gradient elution 10-40% EtOAc/Hexane) to the compound asa white fluffy solid (2.01 g, 86%). ¹H NMR (CDCl₃, 400 MHz) δ=7.80-8.12(m, 10H), 7.60-7.78 (m, 4H), 7.18-7.60 (m, 21H), 6.20-6.05 (m, 2H),5.60-5.91 (m, 5H), 5.10-5.43 (m, 3H), 3.80-5.02 (m, 7H), 3.40-3.56 (m,1H), 1.95-2.10 (m, 4H), 1.00-1.60 (m, 11H). ¹³C NMR (CDCl₃, 100 MHz)δ=169.89, 166.51, 166.40, 166.35, 166.32, 166.24, 166.10, 166.03,165.99, 165.96, 165.86, 165.61, 165.46, 166.38, 165.34, 165.27, 165.23,163.68, 139.36, 133.71, 133.67, 133.56, 133.40, 133.27, 133.21, 130.12,130.05, 129.98, 129.95, 129.92, 129.88, 129.80, 129.77, 129.73, 129.68,129.62, 129.55, 129.50, 129.47, 129.41, 129.40, 129.29, 129.14, 129.11,129.03, 128.96, 128.87, 128.84, 128.83, 128.78, 128.76, 128.63, 128.56,128.54, 128.48, 128.37, 128.26, 114.33, 114.26, 100.92, 100.84, 97.04,96.52, 75.36, 75.17, 74.84, 73.37, 72.95, 72.90, 72.81, 72.57, 72.507,71.94, 71.58, 71.05, 70.37, 70.27, 70.19, 70.06, 69.86, 69.24, 69.19,69.02, 63.71, 63.56, 63.20, 62.93, 62.69, 33.96, 33.91, 32.93, 29.60,29.53, 29.50, 29.46, 29.42, 29.33, 29.30, 29.22, 29.14, 29.06, 29.00.MS. Molecular weight calculated for C₇₁H₆₈O₁₈, Cal. 1208.44. Found1231.4 (M+Na).

Preparation of 219:

Compound 218 (7.26 g, 6 mmol) was dissolved in a mixture of Dioxane:Water (100 mL, 3:1). To the reaction mixture lutidine (0.7 mL, 2 eq),followed by OsO₄ solution (5 mL. 0.05M solution in ^(t)Butanol) wereadded. Sodium periodate (5.11 g, 4 eq) was added and stirred for 4 hr'sat room temperature. Reaction was monitored by TLC, once the startingmaterial was consumed; the mixture was diluted with water and extractedwith DCM (3 times) and dried over sodium sulfate. All the solvents wereremoved and the residue was directly used next reaction. Residue fromthe above reaction was dissolved in DMF (60 mL) to that Oxone (3.86 g,1.05 eq) and stirred at ambient temperature for 3 h. Once the startingmaterial was consumed, 10 mL of 1M HCl was added and diluted with Ethylacetate. Washed with water, brine and dried over sodium sulfate.Solvents were removed and the residue was purified by chromatography(gradient elution 20-40% EtOAc/hexane) to get the compound 219 as awhite solid (5.50 g 75%). ¹H NMR (DMSO-d₆, 400 MHz) δ=12.00 (bs, 1H),8.42-7.10 (m, 35H), 6.10-4.5 (m, 13H), 4.20-3.30 (m, 3H), 2.20-2.03 (m,3H), 1.50-0.8 (11H). ¹³C NMR (DMSO-d₆, 100 MHz) δ=174.55, 174.51,169.13, 165.59, 165.52, 165.39, 165.27, 165.24, 165.14, 164.99, 164.88,164.75, 164.70, 164.66, 164.60, 164.54, 164.50, 162.92, 165.59, 165.51,165.39, 165.27, 165.24, 165.14, 164.99, 164.88, 164.75, 164.70, 164.60,164.54, 164.50, 133.80, 133.71, 133.58, 133.42, 133.29, 133.15, 129.88,129.42, 129.36, 129.29, 129.23, 129.20, 129.12, 129.07, 129.05, 129.03,128.91, 128.88, 128.72, 128.59, 128.48, 128.38, 99.96, 99.29, 99.22,95.96, 95.64, 95.22, 93.10, 75.61, 74.86, 74.57, 74.37, 74.15, 73.59,73.14, 72.58, 71.46, 71.15, 70.48, 70.31, 70.09, 69.97, 69.00, 68.87,68.22, 67.81, 63.65, 62.49, 60.73, 59.76, 43.01, 33.68, 33.62, 32.54,28.84, 28.82, 28.61, 28.55, 28.47, 28.40, 25.47, 25.21, 24.52, 24.43,20.45. MS. Molecular weight calculated for C₇₀H₆₆O₂₀, Cal. 1226.41.Found 1249.4 (M+Na).

Preparation of 220:

Compound 219 (1.65 g, 1.37 mmol), hydroxyl proline amine (0.945 g, 1.3eq) and HBTU (0.623 g, 1.64 mmol) were dissolved in DMF under argon.DIEA (0.71 mL, 3 eq.) was added to that and stirred for 3 hrs at ambienttemperature. The reaction was monitored by TLC; once the startingmaterial was consumed the mixture was poured in to an ice water mixture;extracted with ethyl acetate washed with water, brine and dried oversodium sulfate. Solvents was removed and the residue was purified bychromatography (first ethyl acetate followed by a gradient elution 3-10%MeOH/EtOAc) to get the product 220 as a pale yellow solid (1.55 g, 65%).¹H NMR (DMSO-d₆, 400 MHz) δ=8.20-7.32 (m, 35H), 7.32-7.10 (m, 9H),6.90-6.82 (m, 4H), 6.00-5.63 (m, 4H), 5.41-5.37 (m, 1H), 5.20-5.03 (m,2H), 4.98 (d, J=4.15 Hz, 1H), 4.90 (d, J=4.15 Hz, 1H), 4.88-4.05 (m,9H), 3.70 (s, 6H), 3.65-2.93 (m, 10H), 2.20-0.80 (m, 22H). ¹³C NMR(DMSO-d₆, 100 MHz) δ=171.81, 170.94, 170.90, 170.84, 165.56, 165.53,165.49, 165.19, 165.12, 164.87, 164.72, 164.63, 164.58, 164.46, 158.09,158.03, 157.96, 145.08, 144.74, 135.87, 135.73, 135.48, 135.42, 133.80,133.57, 133.42, 133.29, 129.60, 129.55, 129.26, 129.20, 129.04, 129.00,128.87, 128.74, 128.69, 128.59, 128.36, 128.34, 128.27, 128.02, 127.86,127.77, 127.57, 126.74, 126.56, 113.19, 113.09, 99.26, 95.94, 85.77,85.10, 74.83, 73.58, 72.55, 71.43, 70.44, 70.07, 69.01, 68.87, 68.58,68.19, 67.45, 65.19, 63.29, 63.48, 63.33, 62.47, 59.75, 55.59, 54.99,54.96, 53.44, 44.56, 38.21, 36.30, 35.76, 35.41, 34.15, 32.52, 30.74,30.15, 29.09, 28.84, 28.66, 28.56, 28.52, 26.18, 25.27, 25.22, 24.54,24.14. 21.22, 20.75, 20.71, 18.59, 14.07, 13.54 MS. Molecular weightcalculated for C₁₀₂H₁₀₄N₂O₂₄, Cal. 1740.70. Found 1263.7 (M+Na).

Preparation of Long Alkyl Chain CPG 221:

Hydroxy derivative 220 (1.50 g, 0.862 mmol) was dissolved in DCM (20 mL)to that Succinic anhydride (0.174 g, 2 eq) and DMAP (0.316 g, 3 eq.)were added and stirred overnight. TLC showed completion of reaction. Thereaction mixture was diluted with DCM (20 mL), washed successively withcold dilute citric acid and water (2 times), dried over sodium sulfate.Solvents were removed and dried under high vacuum to get the succinate.The succinate from the above step and HBTU (0.392 g, 1.2 eq) weredissolved in DMF (30 mL). DIEA (0.450 mL) was added to that and themixture stirred for 5 minutes under argon. Long chain alkyl amine-CPG(lcaa CPG, 5.30 g, 133 μmol/g) was added to the mixture and gentlyshaken for 2 h. The CPG was filtered, successively washed with DMF, amixture of DCM/MeOH, DCM and dried. The dried CPG was transferred intoanother flask treated with Ac₂O in pyridine (25%) in the presence of TEA(1 mL) for 15 min. under gentle shaking. Finally the CPG was filtered,washed with DCM, DCM:MeOH (9:1), followed by DCM and ether. The CPG 221was dried under vacuum overnight and the loading was measured asreported (5.62 g, loading: 42 μmol/g).

Hydroxy derivative 220 (0.200 g, 0.115 mmol) was dissolved in anhy. DCM(5 mL) to that DIEA (0.80 mL) and chloroamidite reagent (0.068 mL) wasadded and stirred overnight. The reaction was monitored by TLC, solventswere removed under reduced pressure and charged directly charged to asilica gel column (neutralized with TEA). First eluted with 2:1(EtOAc/Hexane) followed by EtOAc to get the product (0.150 g, 67%). ¹HNMR (CDCl₃, 400 MHz) δ=7.10-8.12 (m, 48H), 6.85-6.75 (m, 4H) 6.10 (t,J=10.19 Hz, 1H), 5.80-5.60 (m, 3H), 5.33-5.20 (m, 2H), 5.00-4.06 (m,12H), 3.77 (s, 6H), 3.90-3.05 (m, 16H), 2.80-1.01 (27H). ³¹P(CDCl₃, 161MHz) δ=145.83, 145.41, 144.95 MS. Molecular weight calculated forC₁₁₁H₁₂₁N₄O₂₅, Cal. 1940.81. Found 1963.80 (M+Na).

Example 12. RNA Synthesis and Duplex Annealing

1. Oligonucleotide Synthesis:

All oligonucleotides were synthesized on an AKTAoligopilot synthesizeror an ABI 394 synthsizer. Commercially available controlled pore glasssolid support (dT-CPG, 500 {acute over (Å)}, Prime Synthesis) and RNAphosphoramidites with standard protecting groups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis unless otherwise specified. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcarbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of asequence was achieved by starting the synthesis with the correspondingcarbohydrate solid support. Similarly a cholesterol moiety at the 3′-endwas introduced by starting the synthesis on the cholesterol support. Ingeneral, the ligand moiety was tethered to trans-4-hydroxyprolinol via atether of choice as described in the previous examples to obtain ahydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety wasthen coupled to a solid support via a succinate linker or was convertedto phosphoramidite via standard phosphitylation conditions to obtain thedesired carbohydrate conjugate building blocks. See Examples 1-11 fordetails. Fluorophore labeled siRNAs were synthesized from thecorresponding phosphoramidite or solid support, purchased from BiosearchTechnologies. The oleyl lithocholic (GalNAc)₃ polymer support made inhouse at a loading of 38.6 μmol/gram. The Mannose (Man)₃ polymer supportwas also made in house at a loading of 42.0 μmol/gram.

Conjugation of the ligand of choice at desired position, for example atthe 5′-end of the sequence, was achieved by coupling of thecorresponding phosphoramidite to the growing chain under standardphosphoramidite coupling conditions unless otherwise specified. Anextended 15 min coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate was introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent The cholesterol phosphoramidite was synthesized in house, andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite was 16 minutes.

Syntheses of 3′-Cholesterol-3′-Carbohydreate containing oligonucleotideswas accomplished by coupling of the cholesterol phosphoramidite to thedesired carbohydrate bearing solid support followed by coupling of thenucleoside phosphoramdites. PEGylated Oligonucleotides with or without asecond ligand was obtained by post-synthetic conjugation of thecorresponding PEG-NHS ester to amino-linked sequence. The amino linkerwas introduced at desired position in a sequence by using acorresponding trans-4-hydroxyprolinol based amino linker or commerciallyavailable amino linkers. For example, syntheses of 3′-PEG-3′-GalNAccontaining oligonucleotides was accomplished by coupling oftrans-4-hydroxyprolinol-amino linker phosphoramidite to the desiredGalNAc bearing solid support followed by coupling of the nucleosidephosphoramdites. The oligonucleotide thus obtained was subjected topost-synthetic conjugation with PEG-NHS ester between pH 7.5 and 9 insodium bicarbonate buffer depends on the nature of the sequence.

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 80 mLof a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at55° C. The bottle was cooled briefly on ice and then the ethanolicammonia mixture was filtered into a new 250 ml bottle. The CPG waswashed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume ofthe mixture was then reduced to ˜30 ml by roto-vap. The mixture was thenfrozen on dry ice and dried under vacuum on a speed vac.

3. Deprotection-II (Removal of 2′ TBDMS Group)

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

4. Analysis

The oligonucleotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

5. PEGylation of Sugar Conjugated Oligonucleotides

Oligonucleotide containing functionalized with an amino linker wastreated with PEG-NHS ester of desired molecular weight in sodiumbicarbonate buffer between pH 7.5 and 9.0. The progress of the reactionwas monitored by HPLC. After completion of the reaction the PEGylatedoligonucleotide was purified by HPLC and analyzed by MS.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides werediluted in water to 150 μl and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. siRNA Preparation

For the preparation of siRNA, equimolar amounts of sense and antisensestrand were heated in 1×PBS at 95° C. for 5 min and slowly cooled toroom temperature. Integrity of the duplex was confirmed by HPLCanalysis.

TABLE 2 GalNAc Conjugated duplexes Duplex SEQ ID Target ID No. S/ASSequence 5′-3′ PCSK9 AD-3672 23 A-30693 GccuGGAGuuuAuucGGAAdTdTsL96 24A-18242 PUUCCGAAUAAACUCCAGGCdTsdT PCSK9 AD-3673 25 A-30693GccuGGAGuuuAuucGGAAdTdTsL96 26 A-30696PuUfcCfgAfaUfaAfaCfuCfcAfgGfcdTdTsL10 PCSK9 AD-3674 27 A-30694GccuGGAGuuuAuucGGAAdTdTsQ11L96 28 A-18242 PUUCCGAAUAAACUCCAGGCdTsdTPCSK9 AD-3718 29 A-30983 GccuGGAGuuuAuucGGAAdTdTsL101 30 A-18242PUUCCGAAUAAACUCCAGGCdTsdT PCSK9 AD-3627 31 A-30824GccuGGAGuuuAuucGGAAdTdTL96 32 A-18242 PUUCCGAAUAAACUCCAGGCdTsdT PCSK9AD-3628 33 A-30824 GccuGGAGuuuAuucGGAAdTdTL96 34 A-30682PuUfcCfgAfaUfaAfaCfuCfcAfgGfcdTdTL43 PCSK9 AD-3629 35 A-16865GccuGGAGuuuAuucGGAAdTsdT 36 A-18242 PUUCCGAAUAAACUCCAGGCdTsdT PCSK9AD-3671 37 A-16865 GccuGGAGuuuAuucGGAAdTsdT 38 A-30693GccuGGAGuuuAuucGGAAdTdTsL96 apoB AD-6490 39 A-52965′-GGAAUCuuAuAuuuGAUCcAsA 40 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoBAD-5544 41 A-5474 GGAAUCuuAuAuuuGAUCcAAsL10 42 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB AD-3697 43 A-30863GGAAUCuuAuAuuuGAUCcAAsL96 44 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoBAD-3698 45 A-30864 GGAAUCuuAuAuuuGAUCcAAsQ11L96 46 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB AD-3699 47 A-30863GGAAUCuuAuAuuuGAUCcAAsL96 48 A-30865 uuGGAUcAAAuAuAAGAuUCccsUsL10 apoBAD-3717 49 A-30982 GGAAUCuuAuAuuuGAUCcAAsL101 50 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB AD-18117 51 A-5474GGAAUCuuAuAuuuGAUCcAAsL10 52 A-31849 Q38uuGGAUcAAAuAuAAGAuUCcscsU apoBAD-18118 53 A-30863 GGAAUCuuAuAuuuGAUCcAAsL96 54 A-31849Q38uuGGAUcAAAuAuAAGAuUCcscsU apoB AD-18119 55 A-30864GGAAUCuuAuAuuuGAUCcAAsQ11L96 56 A-31849 Q38uuGGAUcAAAuAuAAGAuUCcscsUapoB Ad-18648 57 A-31644 GGAAUCuuAuAuuuGAUCcAAsQ11L90 58 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB AD-18649 59 A-31649GGAAUCuuAuAuuuGAUCcAAsQ51Q11L96 60 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoBAD-18650 61 A-32147 GGAAUCuuAuAuuuGAUCcAAsQ11L80 62 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB AD-18651 63 A-32148Q11-GGAAUCuuAuAuuuGAUCcAAsL96 64 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoBAD-18652 65 A-32801 GGAAUCuuAuAuuuGAUCcAAsQ11L110 66 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB 67 A-34132 GGAAUCuuAuAuuuGAUCcAAsQ8L11068 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoB 69 A-34133GGAAUCuuAuAuuuGAUCcAAsQ90L110 70 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoB71 A-34134 Q8GGAAUCuuAuAuuuGAUCcAAsL110 72 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB 73 A-34135 Q90GGAAUCuuAuAuuuGAUCcAAsL11074 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoB AD-19031 75 A-33593GGAAUCuuAuAuuuGAUCcAAsQ11L117 76 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoB77 A-34176 GGAAUCuuAuAuuuGAUCcAAsL117 78 A-5475uuGGAUcAAAuAuAAGAuUCcscsU apoB 79 A-32800 GGAAUCuuAuAuuuGAUCcAAsL110 80A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoB 81 A-34156GGAAUCuuAuAuuuGAUCcAAsL82 82 A-5475 uuGGAUcAAAuAuAAGAuUCcscsU apoB 83A-34157 GGAAUCuuAuAuuuGAUCcAAsL83 84 A-5475 uuGGAUcAAAuAuAAGAuUCcscsUFVII AD-18572 85 A-31843 GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsL96 86A-31848 Q11GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-18567 87 A-31844GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ51Q11L96 88 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-18568 89 A-31845GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ11L90 90 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-18569 91 A-31846GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ11L80 92 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-18570 93 A-31847Q11GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsL96 94 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-18571 95 A-32817GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ11L110 96 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII 97 A-35052GGAUCAUCUCAAGUCUUACdTsdTsL10 98 A-4724 GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdTFVII 99 A-33571 GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsL116 100 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII 101 A-33572GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ92L96 102 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII 103 A-4639 GGAUCAUCUCAAGUCUUACdTdT104 A-4640 GUAAGACUUGAGAUGAUCCdTdT FVII 105 A-34128GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ8L110 106 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII 107 A-34129GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ90L110 108 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII 109 A-34130Q8GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsL110 110 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII 111 A-34131Q90GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsL110 112 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-19032 113 A-33573GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ11L117 114 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-19033 115 A-33570GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ91L96 116 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT FVII AD-18047 117 A-31841GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTdTsQ11L96 118 A-4724GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT Note: S is PS linkge, lowercase is2′-O-methyl nucleotide, Nf is 2′-fluoro nucleotide, P is a phosphategroup, L10 is N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol(Hyp-C6-Chol), L43 is Quasar 570 CPG (BG5-5063, Biosearch Tech), L80 isN-[tris(GalNAc-alkyl)-amidohexanoylcarboxamidoethyl-dithio-butyryl]-4-hydroxyprolinol(Hyp-S-S-(GalNAc-alkyl)3), L82 is PEG 5K CarboxymethylNHS, L83 is PEG20K CarboxymethylNHS, L96 isN-[tris(GalNAc-alkyl)-amidodecanoy1)]-4-hydroxyprolinol(Hyp-(GalNAc-alkyl)3), L110 isN-[N′,N″-(bis(GalNAc-alkyl)-lysine)-aminocapryl]-4-hydroxyprolinol(Hyp-Lys-(GalNAc-alkyl)2), L101 is Hyp-(GalNAc-TEG)3-LCO, L116 isN-(lithocholylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-lithocholicacid), Q8 is N-(aminocaproyl)prolinol-4-phosphate, Q11 isN-(cholesterylcarboxamidocaproyl)prolinol-4-phosphate, Q38 is Quasar 570phosphate (BNS-5063, Biosearch Tech), Q90 isN-(PEG(20K)pentylcarboxamidocaproyl)-4-hydroxyprolinol, Q91 isN-(myristylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-C14), Q92 isN-(lithocholylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-lithocholicacid), Q51 is6-hydroxyhexyldithiohexylphosphate (Thiol-Modifier C6 S-SGlen Res. 10-1936) and L117 isN-[N′,N″-(bis(glucose-alkyl)-lysine)-aminocapryl]-4-hydroxyprolinol(Hyp-Lys-(Gluc-alkyl)2).

TABLE 3 Conjugated single strands SEQ ID Alnylam No No Project sequencecalc. MW obs. MW ALSQ-3465 119 ApoB GUCAUCACACUGAAUACCAAUsL36 7512.07574.3 ALSQ-3466 120 ApoB Q11GUCAUCACACUGAAUACCAAUsL36 8216.0 8279.2ALSQ-3467 121 ApoB GUCAUCACACUGAAUACCAAUQ11sL36 8216.0 8279.2 ALSQ-3613122 Eg5 oCoUGAAGAoCoCoUGAAGAoCAAoUdTdTsL49 7587.2 7586.1 ALSQ-3617 123Eg5 oCoUGAAGAoCoCoUGAAGAoCAAoUdTdTsQ38Q49 8360.0 8358.9 ALSQ-3618 124Luc CUUACGCUGAGUACUUCGAdTdTsL49 7395.8 7394.9 ALSQ-3619 125 LucQ38CUUACGCUGAGUACUUCGAdTdTsL49 8016.0 8014.4 ALSQ-31013 126 5′cuGGcuGAAuuucAGAGcAdTdT-(Man)3 3′ Note: oN is 2′-O-methyl ucleotide,lowercase is 2′-F nucleotide, s is PS linkage, L36 is galactose moietyderived from support 207, L49 is maltose moiety derived from support221, Q11 is cholesterol-hydroxyprolinol moiety, Q38 is maltose moietyderived from phosphoramidite 222, (MAN)₃ is a trivalent mannoseconjugate at 3′-end

Example 13. Animal Testing in Mice

Bolus dosing of formulated siRNAs in C57/BL6 mice (5/group, 8-10 weeksold, Charles River Laboratories, MA) was performed by low volume tailvein injection using a 27 G needle. For AD-3629, AD-3671, AD-3672,AD-3673 and AD-3674 dosing was carried out on three consecutive days at100 mg/kg. Mice were kept under an infrared lamp for approximately 3 minprior to dosing to ease injection. 48 hour post last dose mice weresacrificed by CO₂-asphyxiation. 0.2 ml blood was collected byretro-orbital bleeding and the liver was harvested and frozen in liquidnitrogen. Serum, livers and ileums were stored at −80° C. Total serumcholesterol in mouse serum was measured using the Wako Cholesterol Eenzymatic colorimetric method (Wako Chemicals USA, Inc., Richmond, Va.,USA) according to manufacturer's instructions. Measurements were takenon a VERSA Max Tunable microplate reader (Molecular Devices, Sunnyvale,Calif.) using SoftMax Pro software. Message levels of the target geneApoB were measured via bDNA analysis as below.

bDNA analysis:

Frozen livers and ileums were grinded using 6850 Freezer/Mill CryogenicGrinder (SPEX CentriPrep, Inc) and powders stored at −80° C. untilanalysis. PCSK9 mRNA levels were detected using the branched-DNAtechnology based QuantiGene Reagent System (Panomics, Fremont, Calif.,USA) according to the protocol. 10-20 mg of frozen liver powders waslysed in 600 ul of 0.3 ug/ml Proteinase K (Epicentre, #MPRK092) inTissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. forovernight. Then 10 ul of the lysates were added to 90 ul of LysisWorking Reagent (1 volume of stock Lysis Mixture in two volumes ofwater) and incubated at 55° C. overnight on Panomics capture plates withprobe sets specific to mouse PCSK9 and mouse GAPDH (Panomics, USA).Capture plates then were processed for signal amplification anddetection according to the protocol and chemiluminescence was read asrelative light units (RLUs) on a microplate luminometer Victor2-Light(Perkin Elmer). The ratio of PCSK9 mRNA to GAPDH mRNA in liver and ileumlysates was averaged over each treatment group and compared to a controlgroup treated with PBS

Results:

As shown in Table 6, as compared to the PBS control, treatment withcompounds AD-3673, and AD-3674 resulted in significant (˜50%) and (˜76%)lowering of PCSK9 transcript levels in mouse liver and ileum (asindicated by a smaller PCSK9 to GAPDH transcript ratio when normalizedto a PBS control group), indicating that the conjugated siRNA moleculeswere active in vivo. As shown in Table 4, the silencing activitytranslated in lowering of total cholesterol by 32 and 46% respectivelyin those animals.

TABLE 4 Efficacy of GalNAc PCSK9 conjugates in mice. Efficacy of GalNAcPCSK9 conjugates in mice C57BL6 N = 6/group 3 × 100 mg/kg, Sac 1 daypost last injection All data normalized to PBS control Sense AntisenseLiver Ileum Serum strand strand PCSK9/GAPDH SD PCSK9/GAPDH SDCholesterol SD PBS 1.00 0.22 1.00 0.20 1.00 0.07 AD-3629 A-16865 A-182420.73 0.14 1.09 0.24 0.89 0.05 AD-3671 A-16865 A-30696 0.90 0.24 1.080.29 0.70 0.06 AD-3672 A-30693 A-18242 0.86 0.22 0.95 0.13 0.91 0.08AD-3673 A-30693 A-30696 0.50 0.07 0.98 0.13 0.68 0.05 AD-3674 A-30694A-18242 0.24 0.07 0.42 0.17 0.54 0.04

Example 14. Silencing Activity of Cholesterol-(GalNAc)3 ConjugatedsiRNAs Relative to Cholesterol Only Conjugated siRNAs

Bolus dosing of formulated siRNAs in C57/BL6 mice (3/group, 8-10 weeksold, Charles River Laboratories, MA) was performed by low volume tailvein injection using a 27 G needle. Dosing was carried out on threeconsecutive days at 100 mg/kg. Mice were wither sacrificed 24 hour postlast dose and organs harvested and frozen in liquid nitrogen or bloodwas withdrawn on days 1, 2, 5, 8, 11 and 15 post last dose. Harvestedserum, livers and ileums were stored at −80° C. Total serum cholesterolwas measured using the Modified Trinder Methodology Cholesterol Testfrom Stanbio Laboratory (Boerne, Tex., USA) according to manufacturer'sinstructions. Measurements were taken on a VERSA Max Tunable microplatereader (Molecular Devices, Sunnyvale, Calif.) using SoftMax Prosoftware.

bDNA analysis:

Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder(SPEX CentriPrep, Inc) and powders stored at −80° C. until analysis.ApopB and GAPDH mRNA levels were detected using the branched-DNAtechnology based QuantiGene Reagent System (Panomics, Fremont, Calif.,USA) according to the protocol. 10-20 mg of frozen liver powders waslysed in 1000 ul of 0.3 ug/ml Proteinase K (Epicentre, #MPRK092) inTissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 40minutes. Then 10 ul of the lysates were added to 90 ul of Lysis WorkingReagent (1 volume of stock Lysis Mixture in two volumes of water) andincubated at 55° C. overnight on Panomics capture plates with probe setsspecific to mouse ApoB and mouse GAPDH (Panomics, USA). Capture platesthen were processed for signal amplification and detection according tothe protocol and chemiluminescence was read as relative light units(RLUs) on a microplate luminometer Victor2-Light (Perkin Elmer). Theratio of ApoB mRNA to GAPDH mRNA in liver lysates was averaged over eachtreatment group and compared to a control group treated with PBS

Results:

As shown in FIG. 31, as compared to the Cholesterol conjugated siRNA,treatment with cholesterol-(GalNAc)3 conjugated siRNA resulted insignificant lowering of ApoB transcript levels (˜65% vs ˜10%, asindicated by a smaller ApoB to GAPDH transcript ratio when normalized toa PBS control group), indicating that the cholesterol-(GalNAc)3conjugated siRNAs have superior knockdown compared to just thecholesterol conjugated siRNAs. The silencing activity translated inlowering of total cholesterol by ˜50% and ˜90% respectively forcholesterol only and cholesterol-(GalNAc)3 conjugated siRNAs as comparedto PBS control.

As shown in FIG. 32, the cholesterol-(GalNAc)3 conjugated siRNA(AD-3698) showed improved and longer duration of lowering of totalcholesterol than cholesterol only conjugated siRNAs (AD-5544), ˜15 daysversus ˜10 days.

TABLE 5 Sequences for comparison of cholesterol conjugated andcholesterol-(GalNAc)3 conjugated siRNAs. Strand SEQ ID Duplex # Strand #Type NO: Sequence 18117 5474 Sense 127 GGAAUCuuAuAuuuGAUCcAAsQ11 31849AntiSense 128 Q38uuGGAUcAAAuAuAAGAuUCcscsU 18118 30863 Sense 129GGAAUCuuAuAuuuGAUCcAAsL96 31849 AntiSense 130Q38uuGGAUcAAAuAuAAGAuUCcscsU 18119 30864 Sense 131GGAAUCuuAuAuuuGAUCcAAsQ11L96 31849 AntiSense 132Q38uuGGAUcAAAuAuAAGAuUCcscsU 3698 30864 Sense 133GGAAUCuuAuAuuuGAUCcAAsQ11L96 5475 AntiSense 134uuGGAUcAAAuAuAAGAuUCcscsU 5544 5474 Sense 135 GGAAUCuuAuAuuuGAUC cAAsQ115475 AntiSense 136 uuGGAUcAAAuAuAAGAuUCcscsU Lower case lettersrepresent 2′-O-Me modified nucleotides; Chol is cholesterol, L96 isN-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3; Q11 isN-(cholesterylcarboxamidocaproyl)prolinol-4-phosphate, s isphosphorothioate linkage, Q38 is Quasar-570 (Cy3 dye).

Example 15. Comparison of Uptake of Cy3 Labeled siRNA with CholesterolConjugated siRNA Versus Cholesterol-(GalNAc)3 Conjugated siRNA

Bolus dosing of Cy3-labeled siRNAs in C57/BL6 mice (3/group, 16-19 grambody weight, Charles River Laboratories, MA) was performed by tail veininjection. Mice were kept under an infrared lamp for approximately 3 minprior to dosing to ease injection. AD-18117, AD-18118, AD-18119, and PBSdosing was carried out by one single bolus injection at 100 mg/kg. 15minutes or 3 hour post dose mice were anesthetized with avertin (240mg/kg), and then perfused with 4% paraformaldehyde/phosphate-bufferedsaline. The mouse livers were fixed in 4% paraformaldehyde overnight andthen in 20% sucrose/phosphate-buffered saline overnight. Tissues werethen embedded in O.C.T. compound (Tissue-Tek Optimal Cutting TemperatureCompound; Sahura, Torrance, Calif.) and sections were cut at 6 μm with acryostat maintained at −20° C. The slides were analyzed using Carl ZeissAxioVision microscopy. As shown in FIG. 32, cholesterol-(GalNAc)3conjugated siRNA (AD-18119) had superior celloular uptake relative to acholesterol only (AD-18117) or (GalNAc)3 only (AD-1188) conjugatedsiRNAs.

Example 16. In Vivo Silencing of FVII with Carbohydrate ConjugatedsiRNAs Experimental Design is Shown in FIG. 39A

FIGS. 39A-39C and FIGS. 40 (A and B) show the results of in vivosilencing of FVII with carbohydrate conjugated siRNAs.

Example 17. Effect of Spacer, Linkage, Valency and Cholesterol Positionon In Vivo Silencing with Carbohydrate Conjugated siRNAs

Experimental Design

-   -   100 mg/kg (in PBS), i.v. (bolus) once daily for 3 consecutive        days    -   Sac. 24 h after last dose    -   bDNA assay of ApoB (normalized to GAPDH) in liver and jejunum        samples    -   total cholesterol in liver was also measured

Results are show in FIGS. 41A-41B. Conjugates with a disulfide linkageshowed similar inhibition of ApoB levels as with a cholesterol conjugatealone. This was lower than the inhibition seen with conjugates that didnot have the disulfide linkage. This effect was seen regardless of wherethe disulfide linkage was placed. There was a clear preference forplacement of cholesterol on the 3′-end of sense strand as whencholesterol was placed at 5′-end of sense strand a lowering ofinhibition to cholesterol conjugate only levels was seen. Bivalentconjugates were as effective as the trivalent conjugates.

Example 18. Role of Ligand on In Vivo Gene Silencing (GalNAc Vs.Glucose)

Bivalent GalNAc and glucose conjugates (shown below) were used toconfirm involvement of receptor targeting with GalNAc conjugates.

Experimental Design:

100 mg/kg in PBS), i.v. (bolus) once daily for 3 consecutive days

-   -   Sac. 24 h after last dose    -   bDNA of ApoB (normalized to GAPDH) in liver and jejunum samples    -   total liver cholesterol levels also measured

FIGS. 42 (A and B) shows the results. In the liver, GalNAc conjugateshowed a higher inhibition of ApoB than the glucose conjugate orcholesterol only conjugate. However in the jejunum all three conjugatesshowed similar activity. As there are no ASGP-R1 in jejunum, activityseen could have been due to the presence of cholesterol in all threedesigns.

Example 19. Silencing Activity of Carbohydrate Conjugates In Vitro

Primary mouse hepatocytes were seeded in collagen coated 6-well dishesfor either 1 or 6 days to down regulate the ASGPR. 5 mM CaCl₂ was usedto activate the ASGR. ApoB or Luc siRNAs were added at 2 uM in serumfree media and uptake allowed to proceed for 24 hours. Cells were lysedand ApoB mRNA knockdown evaluated by bDNA assay. A western blot was doneas a control to confirm ASGR downregulation at day 6. FIG. 43 shows thatall three conjugates (cholesterol alone, GalNAc alone andcholesterol-GalNac together) showed ApoB mRNA silencing at day 1. Afterseveral days in culture, ability of GalNAc conjugates to silence isimpaired consistent with downregulation of receptors such as the ASGRknown to occur with extended culture times of primary cells. Cholesterolconjugate also showed some reduction in the ability to silence at day 6.The following siRNAs were used:

AD-1955 (control, −/−)

AD-6490 (control, −/Cy3)

AD-5546 (Chol/Cy3)

AD-3697 (GalNAc/Cy3)

AD-3698 (GalNAc+Chol/Cy3)

Example 20. Competition of Carbohydrate Conjugated siRNAs with ASGRLigand Asilofetuin (ASF) During In Vitro Uptake

Primary mouse hepatocytes were seeded in collagen coated 6-well dishesfor 1 day. 5 mM CaCl₂ was used to activate the ASGR. The binding ofGalNAc conjugated siRNAs was competed with increasing amountsAsialofetuin prior to and during siRNA incubation. ApoB and Luc siRNAswere added at 4 uM in serum free media and uptake allowed to proceed for24 hours. Cells were lysed and ApoB mRNA knockdown evaluated by bDNAassay. FIG. 44 shows that presence of Asilofetuin competed with uptakeof GalNAc and Cholesterol-GalNAc conjugated siRNAs. The ability tooutcompete silencing of ApoB with Asilofetuin suggests an interactionbetween GalNAc and AGSR is important for mediating uptake and activity.The following siRNAs were used:

AD-1955 (control, −/−)

AD-6490 (control, −/Cy3)

AD-3697 (GalNAc/Cy3)

AD-3698 (GalNAc+Chol/Cy3)

Example 21. In Vitro Receptor Binding and Uptake of CarbohydrateConjugates

Primary mouse hepatocytes were seeded in collagen coated 6-well dishesfor either 1 or 6 days to down regulate the ASGPR. 5 mM CaCl₂ was usedto activate the ASGR. ApoB duplexes were added at 1 uM in serum freemedia and uptake allowed to proceed for 6 hours. After uptake wascomplete, cells were fixed in 3.7% PFA and counter stained with DAPI. Ascan be seen in FIG. 45, Cy3 labeled siRNAs comprising either acholesterol conjugate (AD-18117) or a cholesaterol+GalNAc conjugate(AD-18119) were taken up much more effectively than a Cy3 labeled siRNAwithout a conjugate (AD-18560) or with a GalNAc conjugate only(AD-18118).

Example 22. In Vivo ApoB Gene Silencing with Galactose Conjugated siRNAs

Mice were injected by IV bolus at 50 mg/kg. The galactose conjugatedsiRNA shown in FIG. 46A was synthesized from compound 207. FIG. 46 Bshows that siRNA comprising both a cholesterol and galactose conjugateled to gene silencing in comparison to siRNA comprising only thegalactose conjugate.

We claim:
 1. An oligonucleotide comprising one or more conjugatestructures shown below:

wherein: X is CO, NH, O, S, OC(O), NHC(O), or CH₂; Y is absent, NH, O,S, CO, CH₂, C(O)O, C(O)NH,

Z is C(O)NH, NHC(O), C(O)O, OC(O), NHC(O)O, OC(O)NH, NHC(O)NH, S—S, O,NH, NMe, or CH₂;

is absent, —(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)O—, —(CH₂)_(n)S—,O(CH₂CH₂O)_(n)CH₂CH₂OH, abasic sugars, amide, carboxy, amine, oxyamine,oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, orbiotin and fluorescein reagents;

is a cyclic carrier selected from pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and decalin; or

is an acyclic carrier selected from a serinol backbone or diethanolaminebackbone; Sugar is a monosaccharide selected from the group consistingof galactose, galactosamine, N-acetylgalactosamine, mannose, glucose,glucosamine, N-acetyl-glucosamine, fucose, and lactose; n is 1-6; q is0-10; and

represents a bond joining the conjugate structure to theoligonucleotide.
 2. The oligonucleotide of claim 1, wherein

is a hydroxyprolinol-based linker.
 3. The oligonucleotide of claim 2,wherein

has the structure of:


4. The oligonucleotide of claim 1, wherein q is 0, 1, or
 2. 5. Theoligonucleotide of claim 1, wherein the sugar is


6. The oligonucleotide of claim 1, wherein the oligonucleotide is doublestranded.
 7. The oligonucleotide of claim 6, wherein the oligonucleotidecomprises at least one said conjugate structure at the 3′-end of one ofthe strands.
 8. The oligonucleotide of claim 6, wherein theoligonucleotide comprises at least one said conjugate structure at the5′-end of one of the strands.
 9. The oligonucleotide of claim 6, whereinthe oligonucleotide comprises at least two said conjugate structures atboth ends of one of the strands.
 10. The oligonucleotide of claim 6,wherein the oligonucleotide comprises at least one said conjugatestructure at each strand.
 11. The oligonucleotide of claim 1, whereinthe oligonucleotide is single stranded.
 12. The oligonucleotide of claim11, wherein the oligonucleotide comprises at least one said conjugatestructure at the 3′-end of the strand.
 13. The oligonucleotide of claim11, wherein the oligonucleotide comprises at least one said conjugatestructure at the 5′-end of the strand.
 14. The oligonucleotide of claim11, wherein the oligonucleotide comprises at least two said conjugatestructures at both ends of the strand.
 15. A method of modulating theexpression of a target gene in a cell, comprising providing to said cellthe oligonucleotide of claim
 1. 16. A pharmaceutical compositioncomprising the oligonucleotide of claim 1 alone or in combination with apharmaceutically acceptable carrier or excipient.
 17. Theoligonucleotide of claim 2, wherein

has the structure of:

wherein n is 1-6.
 18. The oligonucleotide of claim 17, wherein

has the structure of:


19. The oligonucleotide of claim 4, wherein q is 2, and the conjugatestructure is


20. The oligonucleotide of claim 5, wherein the conjugate structure is

wherein n′ is 3-6.
 21. The oligonucleotide of claim 20, wherein n′ is 3.22. The oligonucleotide of claim 5, wherein the conjugate structure is

wherein n′ is 3-6.
 23. The oligonucleotide of claim 22, wherein n′ is 3.24. The oligonucleotide of claim 5, wherein the conjugate structure is

wherein n′ is 3-6.
 25. The oligonucleotide of claim 24, wherein n′ is 3.26. The oligonucleotide of claim 5, wherein the conjugate structure is

wherein n′ is 3-6.
 27. The oligonucleotide of claim 26, wherein n′ is 3.28. The oligonucleotide of claim 5, wherein the conjugate structure is:


29. The oligonucleotide of claim 5, wherein the conjugate structure is: